PA1-987

antibody from Invitrogen Antibodies

Targeting: GAPDHS GAPD2, GAPDH-2, GAPDS

Provider product page for PA1-987

Western blot

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Antibody data

Antibody Data
Antigen structure
References [54]
Comments [0]
Validations
Western blot [2]
Other assay [30]

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Product number: PA1-987 - Provider product page
Provider: Invitrogen Antibodies
Product name: GAPDH Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-987 detects rat GAPDH. Shares 100% sequence homology with human and mouse.
Concentration: 1 mg/mL

GAPDHS protein structure - PA1-987 shown in red.

Supportive validation

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Experimental details: Western blot analysis of GAPDH was performed by loading 25 µg of Hela (lane 1), 293 (lane 2), HUVEC (lane 3) and PC12 (lane 4) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a GAPDH polyclonal antibody (Product # PA1-987) at a dilution of 1:3000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~36 kDa.

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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), COS-7 (Lane 2), MDCK (Lane 3), PC-3 (Lane 4) and tissue extract (30 µg lysate) of Ms Brain (Lane 5). The blot was probed with Anti-GAPDH Polyclonal Antibody (Product # PA1-987, 1:1,000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 37 kDa band corresponding to GAPDH was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: Figure 3. The octameric right leg subunits eIF3k and eIF3l impact their mutual expression and are dispensable for the integrity of the rest of eIF3, and the octameric right arm subunit eIF3e stabilizes binding of the right leg subunits (k, l) and eIF3d to the octamer, as well as the octamer attachment to the YLC. Protein levels of all eIF3 subunits and other eIFs upon knock down of either eIF3k, l or e were determined by Western blotting ( A, C, E , respectively). GAPDH was used as a loading control. To assess the integrity of eIF3, the anti-eIF3b co-immunoprecipitation experiments were carried out for each knock-down and the immunoprecipitated eIF3 subunits along with eIF3b were detected by Western blotting ( B, D, F ). The octameric eIF3 subunits are arranged at the left-hand side of each panel and non-octameric subunits are at the right-hand side. The YLC subunits are grouped and highlighted in bold. Quantifications of at least five independent experiments with standard deviations are shown in Supplementary Figure S4 and Table 1 .

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Experimental details: Figure 5. G. intestinalis secreted cysteine proteases have effects on epithelial barrier proteins. A, B, C. Levels of tight junction and adherence junction proteins in Caco-2 whole cell extracts were examined after 24 h of recombinant cysteine proteases treatment (A, 14019; B, 16779; C, 16160), respectively, by Western blot analysis, with GAPDH used as a loading control. Barrier proteins were detected with a mouse or rabbit anti-barrier protein antibody and then incubated with a HR- conjugated mouse or rabbit antibody. D E, F. The localization of occludin in Caco-2 cells visualized by immunofluorescence microscopy. Caco-2 monolayers were incubated with 2.5 ug/ml of 14019 (D), 16779 (E), 16160 (F) in the absence or presence of E64 (10 uM) for 24 h, followed by fixation of PFA. Occludin was detected with a mouse anti-occludin antibody and then incubated with an Alexa Fluor 488-conjugated mouse antibody. Images were taken by a Zeiss Axioplan II Imaging fluorescence microscope and analyzed by ZEN 2.1 software. Bar = 50 um.

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Experimental details: Fig. 4 Ethyl acetate biosynthesis in K. marxianus by alcohol- O -acetyltransferase. a RT-qPCR analysis of wild-type K. marxianus ATF transcript levels at lag (5 h), log (10 h), and stationary (18 h) phases. mRNA copy number represents total number of transcripts for 5 ng of isolated RNA. b Km ATF disruption reduces ethyl acetate production in both aerobic and anaerobic conditions. Cultures were grown at 37 degC for 10 h (aerobic) and 14 h (anaerobic). c Lysate Atf activity from S. cerevisiae with overexpressed Km ATF and Sc ATF1. Rates are reported per mg of lysate protein. Enzyme expression was confirmed by Western blot analysis using anti-c-MYC and anti-GAPDH antibodies. Bars and error bars represent the arithmetic mean and standard deviation of triplicate biological samples. Statistical significance ( P < 0.05) is indicated by asterisk and was determined using a T test

Supportive validation

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Experimental details: Fig. 7 Hemiacetal oxidation activity of K. marxianus alcohol dehydrogenases. Km ADH1-7 were heterologously overexpressed in S. cerevisiae BY 4742. Cell lysate assays showed that Km Adh7 exhibits activity toward synthesis of ethyl acetate from hemiacetal. Enzyme expression was confirmed by Western blot using anti-c-MYC and anti-GAPDH antibodies. Bars and error bars represent the arithmetic mean and standard deviation of triplicate samples

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Experimental details: Figure 5 APOA4 activation of PI3K-AKT pathway is partly LRP1-dependent. ( A ) APOA4 promoted phosphorylation of AKT (pAKT) in a time-dependent manner. Representative western blot images are shown. After transfer of proteins, the membranes were cut into three sections before probing with specific antibodies for western blots. The top section (> 75kD) was used for LRP1 detection, the middle sections (50-75 kD) for pAKT and total AKT detection, and the bottom sections (25-50 kD) for GAPDH detection. This was necessary as stripping and re-probing compromises quantification and running separate gels would not allow for quantification. Bar graphs depicting normalized pAKT and AKT levels to GAPDH. Compared to 0 min, the pAKT/AKT ratio was significantly increased by 2.6-fold at 60 min. ( B ) Representative western blot images showing LRP1, pAKT, AKT, and GAPDH. After pAKT detection, the blot was stripped and used for detecting total AKT. At 60 min post APOA4 treatment, when LRP1 levels were reduced by ~ 50% compared with controls, the pAKT/AKT ratio decreased by ~ 58%, suggesting that APOA4-stimulated increases in pAKT are in part, LRP1-dependent (n = 3/group).

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Experimental details: Fig. 3 FHL124 cells block caspase 8, 9, and 3 activation and PARP-1 cleavage. A Activated caspase-8, caspase-9, and caspase-3 were detected in HeLa cells starting at 3 h and further increased at 5 h and 7 h after TNFalpha and CHX stimulation. No activated caspase-8, caspase-9, and caspase-3 were detectable in FHL124 cells spanning 24 h of treatment with TNFalpha and CHX. A higher pro-caspase-9 quantity (around 2-fold) was seen in HeLa cells than in FHL124 cells, but a higher pro-caspase-3 quantity (around 3-fold) was seen in FHL124 compared to that in HeLa cells. B Cleavage PARP-1 was detected only in HeLa cells but not in FHL124 cells after TNFalpha and CHX stimulation.

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Experimental details: Figure 2 Hypoxia/reoxygenation (H/R) induces miR-19b expression and Sdc1 inhibition. ( A , B ) The levels of miR-19b and Sdc1 mRNA were measured by RT-qPCR in HLMECs exposed to either normoxia or hypoxia then reoxygenation at 0, 3 and 6 h. N = 4/group; mean +- SD; ANOVA with Bonferroni. ( C ) Expression of Sdc1 and GAPDH was detected by Western blot.

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Experimental details: Fig. 1 The overproduction of H 2 S in human CRC. A , B H 2 S production measured in human colorectal cancer specimens (* P < 0.05 vs. normal mucosa) and in colorectal cancer cell lines (* P < 0.05 vs. NCM460 cells; n.s. : no significant difference v.s. MCF-10A) by the H 2 S microsensor. (T: tumor tissue; A: aside colorectal tumor tissue; N: normal colorectal tissue). C H 2 S production in different stage of human colorectal cancer specimens measured by the H 2 S microsensor. D Western Blot of CBS protein expression in human colorectal cancer specimens, paired with the adjacent tissue and corresponding normal mucosa tissues. Membranes are re-probed for GAPDH expression to show that similar amounts of protein were loaded in each lane. E Immunohistochemistry of CBS protein expression in human colorectal cancer specimens, paired with the adjacent tissue and corresponding normal mucosa tissues. F Western Blot of CBS protein expression in colon cancer cell lines (vs. NCM460). G H 2 S production is measured in colon cancer cell lines treated by AOAA. H Cell viability of the HCT116 and CT26 treated with AOAA for 24 h. I Cell growth of the HCT116 and CT26 treated with AOAA within 6 days. (* P < 0.05), (n.s. : No significant difference, P value > 0.05)

Supportive validation

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Experimental details: Fig. 3 The H 2 S scavenging and tumor ferroptosis induced by VZnO in CRC. A The GSH detected by total glutathione assay kit in HCT116 and CT26 after VZnO and ID treatments. B Cell viability by MTT assay in HCT116 and CT26 after VZnO, ID, GSH and Fer-1 treated. C Intracellular ROS levels measured by DCFH-DA assay kit in HCT116 and CT26 with or without VZnO and ID treated. D Colocalization of oxidized lipid and nucleus co-stained by BODIPY C11(green) and DAPI (blue) in HCT116 and CT26 cells. E Representative picture of the ultrastructure of mitochondria in HCT116 and CT26 after VZnO and ID treated. F Average number of mesenchymal cristae per mitochondrion in VZnO-treated cells (statistics of 20 mitochondria) G Representative GPX4, COX2, NOX1 and TFR1 protein expression by Western Blot in HCT116 and CT26 with or without VZnO and ID treated. Membranes were re-probed for GAPDH expression to show that similar amounts of protein were loaded in each lane. (* P < 0.05), (n.s. : No significant difference, P value > 0.05)

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Experimental details: Fig. 5 Anti-tumor activity of VZnO in orthotopic CRC model. A Experimental timeline of the orthotopic CRC model. B Bioluminescence imaging of orthotopic colon cancer mouse injected with different treatments on day 14. C , D Photograph and volume of the tumors with different treatments on day 14. E Representative H&E stained of liver and tumor tissue on day 14. F Body weights of orthotopic colon cancer model. G The continuously H 2 S production in mouse model. H Representative GPX4, COX2, NOX1 and TFR1 protein expression by Western Blot in tumor on day 14. Membranes were re-probed for GAPDH expression to show that similar amounts of protein were loaded in each lane. I) H 2 S content measured on day 14. J The volume of the tumors in the orthotopic breast model on day 14. (* P < 0.05), (n.s. : No significant difference, P value > 0.05)

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Experimental details: Fig. 6 Transcriptome analysis in VZnO-treated HCT116. A KEGG pathway analysis based on the RNA-seq results in HCT116. B Representative heatmap of gene expression levels. C Representative scatter plot of 140 significant genes (33 unregulated genes marked in red and 107 downregulated genes marked in green) for Treat vs. Con. D mRNA levels of GGCT, RRM1 and RRM2 by RNA-seq. E Representative Western Blot result of GGCT, RRM1 and RRM2. Membranes were re-probed for GAPDH expression to show that similar amounts of protein were loaded in each lane

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Experimental details: Fig. 1 Huntingtin (HTT)-lowering activity in vitro. a Chemical structures of HTT-C1 and HTT-D1. b Electrochemiluminescence (ECL) analysis of mutant HTT protein from fibroblasts isolated from a homozygous patient with Huntington's disease (HD) (GM04857) after 96 h of continuous treatment with HTT-C1 and HTT-D1 (0.01-1.0 muM). Representative graphs show percent HTT remaining relative to the dimethyl sulphoxide (DMSO) control. Cell viability assays were performed in parallel. Data represent mean of two ( n = 2) biologically independent samples per data point from one dose-response experiment. c Western blot of HTT protein and housekeeping proteins, oxidoreductase-protein disulphide isomerase (PDI), beta-actin, alpha serine/threonine-protein kinase (AKT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in HD fibroblasts after 96 h of continuous treatment with HTT-C1 (0.015-1.0 muM). Utrophin (UTRN) was also used as a loading control. The western blot data used a representative splicing modifier (tested at multiple concentrations) from a single experiment. Multiple splicing modifiers from the same class were tested and evaluated by western blot analyses. d Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of HTT mRNA in patient fibroblasts after 24 h of treatment with HTT-C1 and HTT-D1 (0.01-1.0 muM). Representative graphs show percent HTT mRNA remaining relative to DMSO control; normalised to housekeeping gene, TATA-box binding protein. Data repr

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Experimental details: Fig. 1. NRP-1 is incorporated into HIV-1 virion and is mainly expressed in macrophages and DCs. ( A ) The schematic representation of the experimental design used to identify NRP-1 in HIV-1 virions. ( B and C ) Total RNA was extracted from primary monocytes, MDMs, MDDCs, THP-1, PMA-treated THP-1, stimulated or resting CD4 + T cells, established cell lines 293T cells, HeLa cells, and Jurkat cells. NRP-1 transcript levels were measured using qPCR and then normalized to GAPDH levels ( B ). Meanwhile, Western blotting was conducted to assess the level of NRP-1 and GAPDH ( C ). ( D ) The 293T cells exogenously expressing the vector encoding NRP-1-GFP were visualized by microscopy. (Scale bar, 10 mum.) ( E ) MDMs or MDDCs were lysed, and membrane-bound and cytosolic proteins were separated to assess the levels of NRP-1, Caveolin-1, and GAPDH using Western blotting with specific antibodies. All blotting data are representative of three independent experiments.

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Experimental details: Fig. 3. NRP-1 inhibits HIV-1 infection in macrophages. ( A and B ) Lentiviral shRNA-transduced MDMs were infected with 10 (low-dose) or 100 (high-dose) ng of HIV-1 AD8 for 18 d. Viral production was measured using p24 ELISA at the indicated time points ( A ). The aliquoted cells were lysed for Western blotting to assess the levels of NRP-1 and GAPDH ( B ). ( C and D ) MDMs were transduced with lentiviral shRNA targeting the 3'-UTR of the NRP-1 or control transcript, and the shNRP-1 -depleted MDMs were then transduced with or without a lentiviral expression vector encoding an untagged NRP-1. Next, the MDMs were infected with 10 (low-dose) or 100 (high-dose) ng HIV-1 AD8 for 18 d, and viral production was measured by p24 ELISA at the indicated time points ( C ). The shRNA-transduced MDMs were lysed for Western blotting to assess the levels of NRP-1 and GAPDH ( D ). ( E ) The MDMs transfected with siRNA targeting NRP-1 or the control were infected with HIV-1 AD8 (multiplicity of infection = 1) and washed twice with PBS to remove the input viruses 6 h after infection. At 3 d after infection, 20 ng of the produced HIV-1 virions in the supernatants were used to infect TZM-bl indicator cells to measure viral progeny infectivity. Total RNA was extracted from the MDMs for qPCR to measure NRP-1 transcript levels normalized to GAPDH levels. ** P < 0.01.

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Experimental details: Fig. 4. NRP-1 is incorporated into virions to restrict HIV-1 infection. ( A ) The schematic representation of NRP-1 domains. The full-length NRP-1 possesses a ligand-binding ectodomain consisting of a1/a2 and b1/b2 domains, followed by a membrane-proximal c domain involved in oligomerization. The remainder of the protein comprises a short membrane-spanning region and cytoplasmic tail (cyto). ( B and C ) NRP-1 is incorporated into viral particles to inhibit infectivity. The 293T cells were cotransfected with constructs encoding FLAG-tagged NRP-1 or its mutants and GFP, along with NL4-3 proviral vectors. At 2 d after cotransfection, 100 ng of the produced HIV-1 virions in the supernatants were used to infect TZM-bl indicator cells to measure viral progeny infectivity ( B ), and the cells were lysed for Western blotting to assess the levels of NRP-1, its mutants, Gag, gp120, and GAPDH with their specific antibodies. The produced virion particles in the cell supernatants were enriched by sucrose cushion ultracentrifugation and subsequently isolated using anti-CD44 microbeads with a magnetic-based method. The purified virion was lysed for Western blotting to assess the NRP-1 or its mutants, CA (p24), and gp120 levels ( C ). All blotting data are representative of three independent experiments. ** P < 0.01; ns, not significant.

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Experimental details: Fig. 5. NRP-1 inhibits HIV-1 virion attachment to target cells. ( A and B ) The BlaM-Vpr-based viral entry assay using a VSV-G-pseudotyped HIV-1. The 293T cells were cotransfected with a construct encoding FLAG-tagged NRP-1 or a mock expression construct with or without pNL4-3.GFP.R + E - reporter vectors (VSV-G) combined with an expression construct encoding BlaM-Vpr. Two days after transfection, 50 ng of the produced virions in the supernatants were used to infect stimulated CD4 + T cells. The fluorescence-activated cell sorter dot plots are representative of five independent experiments ( A ). The data of five independent experiments are plotted as mean +- SEM ( B ). *** P < 0.001. ( C ) The HIV-1 attachment assay. The 293T cells were cotransfected with a construct encoding FLAG-tagged NRP-1 or a mock expression construct at different doses, along with HIV-1 NL4-3 proviral vectors. Two days after transfection, TZM-bl reporter cells or stimulated CD4 + T target cells were spinoculated with 500 ng of the produced HIV-1 particles at 25 degC for 2 h and washed with PBS twice to remove the input virus after spinoculation. The cells were further lysed for the p24 ELISA to quantify the bound viral particles. ( D - G ) MDMs ( D and E ) or MDDCs ( F and G ) were cotransfected with siRNA targeting NRP-1 or the control along with HIV-1 AD8 proviral vectors. At 3 d after cotransfection, the cells were lysed for Western blotting to assess the levels of NRP-1 and GAPDH, and 20 ng of the

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Experimental details: 10 Figure Kv7.2 and Kv7.3 subunits expression in superficial and deep layers in the motor cortex of developing KCNQ2 WT/WT and KCNQ2 WT/T274M mice A , western blots of Kv7.2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at postnatal day (PND) 7, 21 and 30 in superficial layers ( Aa ) and deep layers ( Ab ). B , boxplots quantifying the Kv7.2 subunit relative to the housekeeping protein GAPDH in superficial ( Ba ) and deep layers ( Bb ) of the motor cortex from KCNQ2 WT/WT (black dots) and KCNQ WT/T274M mice (blue dots and purple dots) aged 7, 21 and 30 days. Each dot corresponds to quantification in one mouse. C , western blots of Kv7.3 and of GAPDH in both superficial ( Ca ) and deep layers ( Cb ). D , boxplots quantifying the Kv7.3 subunit relative to GAPDH in superficial ( Da ) and deep layers ( Db ) of the motor cortex from KCNQ2 WT/WT (black dots) and KCNQ WT/T274M mice (blue dots and purple dots) aged 7, 21 and 30 days. Statistical analysis for the effect of the variant across age was by two-way ANOVA. The two factors were the genotype and the age. For Ba , interaction P = 0.32, genotype P = 0.38 and age P = 0.005. For Bb , interaction P = 0.83, genotype P = 0.21 and age P = 0.0001. For Da , interaction P = 0.17, genotype P = 0.7 and age P = 0.23. For Db , interaction P = 0.06, genotype P = 0.06 and age P = 0.005. Statistical analysis for the comparisons of wild-type with knock-in (KI) mice at PND 7, 21 or 30 was by Student's unpaired t test and the Mann-Whitney

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Experimental details: Protein expression in murine and human primary fibroblasts. ( A ) Western blots for JAG1, DUSP7, ITGB3 and MAP2K1, each with GAPDH, of three independent replicates (each band represents a pool of three technical triplicates) in PMFs. ( B ) Western blots for JAG1, DUSP7, ITGB3 and MAP2K1, with GAPDH of three independent replicates (each band represents a pool of three technical triplicates) in HUAFs. ( C - L ) Quantifications of protein band intensities, normalized to GAPDH. Data are represented as mean +- SEM. * * P < 0.01, compared with GM-ctrl.

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Experimental details: The protein level of RIP3 is elevated in hypertrophic hearts. (a) Representative Western blots and quantitation of RIP3 expression in heart tissue from patients with dilated cardiomyopathy (DCM) and normal controls. (b) (Up) Western blots of RIP3, ANP, beta -MHC, and GAPDH and (down) relative quantitation of RIP3 expression in heart tissue from rats after sham or AB operation. (c) (Up) Western blots of RIP3, ANP, beta -MHC, and GAPDH and (down) relative quantitation of RIP3 in NRCMs stimulated with Ang-II (1 mu M) or PBS. (d) (up) Western blots of RIP3, ANP, beta -MHC, and GAPDH and (down) relative quantitation of RIP3 in NRCMs stimulated with PE (50 mu M) or PBS. kDa: kilo-Dalton; * P < 0.05, ** P < 0.01, and *** P < 0.001.

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Experimental details: FIG 1 IFN-alpha treatment induces OTOF in macrophages. (A) OTOF was induced in the PBMCs of untreated patients with HIV infection. Volcano plot of genes differentially expressed in PBMCs isolated from five independent healthy donors and five independent untreated patients with HIV-1 infection. The log 2 fold change difference is presented on the x axis, and the negative log of the false-discovery rate (FDR) is presented on the y axis. Each point represents one gene, which had detectable expression in both groups (five untreated patients with HIV-1 infection versus five healthy donors). The significantly differentially expressed genes are plotted in red with upregulated genes on the right side and downregulated genes on the left side. The nonsignificantly differentially expressed genes are shown as black points. (B) Relative expression of OTOF in the PBMCs of healthy donors ( n = 16), patients with HIV-1 who did not receive ART ( n = 26), and patients with HIV-1 who received ART ( n = 20; viral loads of

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Experimental details: FIG 2 IFN-alpha induces OTOF to restrict HIV-1 infection in macrophages. (A and B) Lentiviral shRNA-transduced THP-1 cells (A) and MDMs (B) were pretreated with IFN-alpha (1,000 U/mL). At 24 h after treatment, cells were washed using culture medium and subsequently infected with 100 ng of HIV-1 NL4-3.Luc.R-.E- (VSV-G). At 24 h after infection, cells were lysed to measure luciferase reporter activity and for Western blotting to assess the levels of OTOF and GAPDH. RLU, relative luminescence units. (C) MDMs were transfected with siRNA against OTOF or control siRNA, and cells were treated with IFN-alpha (1,000 U/mL) 6 h after transfection. At 24 h after treatment, cells were washed and further infected with 50 ng of replication-competent HIV-1 AD8 . At 72 h after infection, viral load in culture supernatants was measured via p24 ELISA; cells were lysed for Western blotting to assess the levels of OTOF and GAPDH. *, P < 0.05; **, P < 0.01 (two-tailed, unpaired Student's t test). EFV, efavirenz. Data are plotted as the means +- standard deviations for three triplicates and representative of three independent experiments. All Western blot data are representative of three independent experiments. Numbers at left of blots are molecular masses in kilodaltons.

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Experimental details: FIG 3 OTOF suppresses HIV-1 and HIV-2 infection and slightly suppresses SIV infection but does not suppress EIAV and MLV infection. (A) 293T cells were transfected with a construct encoding FLAG-tagged OTOF or a mock expression construct at the indicated doses. At 24 h after transfection, cells were infected with 10 or 100 ng of HIV-1 NL4-3.Luc.R-.E- (VSV-G). At 24 h after infection, cells were lysed to measure luciferase reporter activity. (B to E) 293T cells were transfected with a construct encoding FLAG-tagged OTOF or a mock expression construct at the indicated doses. At 24 h after transfection, cells were infected with VSV-G pseudotyped luciferase reporter viruses: HIV-2 Rod -luc (B), SIV mac239- luc (C), EIAV-luc (D), or MLV-luc (E). At 24 h after infection, cells were lysed to measure luciferase reporter activity and for Western blotting to assess the levels of OTOF and GAPDH using specific antibodies. **, P < 0.01; *, P < 0.015; n.s., not significant (two-tailed, unpaired Student's t test). Data are plotted as the means +- standard errors of the means for three independent experiments. All Western blot data are representative of three independent experiments. Numbers at left of blots are molecular masses in kilodaltons.

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Experimental details: FIG 4 Antiviral activity of OTOF is independent of Ca 2+ . (A and B) 293T cells were transfected with a construct encoding FLAG-tagged OTOF or a mock expression construct. At 24 h after transfection, cells were infected with 100 ng of HIV-1 NL4-3.Luc.R-.E- (VSV-G) in the presence or absence of Ca 2+ (A) or Mg 2+ (B) at the indicated doses. At 24 h after infection, cells were lysed to measure luciferase reporter activity and for Western blotting to assess the levels of OTOF and GAPDH using specific antibodies. The inhibition of viral infection was accordingly calculated for OTOF and mock. Data are plotted as the means +- standard errors of the means from three independent experiments. (C) 293T cells were transfected with a construct encoding FLAG-tagged OTOF or a mock expression construct. At 24 h after transfection, cells were infected with 100 ng of HIV-1 NL4-3.Luc.R-.E- (VSV-G) in the presence or absence of thapsigargin at the indicated doses. At 24 h after infection, cells were lysed to measure luciferase reporter activity and for Western blotting to assess the levels of OTOF and GAPDH using specific antibodies. Data are plotted as the means +- standard deviations from three triplicates. All Western blot data are representative of three independent experiments. Numbers at left of blots are molecular masses in kilodaltons.

Supportive validation

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Experimental details: FIG 1 Caspase-3 is required for deoxyadenosine-induced killing of macrophages. (A) U937-derived macrophages (MPhi) were treated with (+) or without (-) dAdo, and cell lysates were analyzed for caspase-3 activity using a colorimetric assay. As controls, lysates were treated with the caspase-3 inhibitor Ac-DEVD-CHO (+ Inhib.). (B) Survival of MPhi exposed to dAdo and increasing concentrations of Z-DEVD-FMK (0 to 5 muM), an inhibitor of caspase-3. (C) Diagram illustrating the position of CASP3 on chromosome 4 and exons 1 to 8 of CASP3 mRNA. Sequencing results for mutated exon 5 alleles (red box) cloned from CASP3 -/- cells are shown. (D) Immunoblotting of lysates from wild-type (WT) MPhi and their CASP3 -/- and complemented CASP3 -/- variants (+ CASP3 WT ) with caspase-3 and GAPDH-specific antibodies (alpha-CASP3 and alpha-GAPDH, respectively). GAPDH was used as a loading control. Numbers to the left of blots indicate the migration of molecular weight markers in kilodaltons. (E and F) Survival of MPhi (black bars) and their SLC29A1 -/- (blue bars), CASP3 -/- (red bars), and complemented CASP3 -/- variants (+ CASP3 WT , white bars) after treatment with dAdo (E) or after treatment with culture medium (RPMI 1640) that had been conditioned by incubation with either wild-type S. aureus Newman or its adsA mutant in the presence of host DNA, as indicated by + and - signs (F). All samples received adenosine deaminase inhibitor (50 muM dCF). Data are the mean (+- standard deviation [SD])

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Experimental details: FIG 3 Caspase-3 activity suppresses macrophage infiltration into staphylococcal abscesses. (A to P) Immunohistochemical analysis of renal tissues isolated 5 days after intravenous injection of 10 7 CFU of wild-type S. aureus Newman or its adsA mutant into C57BL/6 CASP3 fl/fl or CASP3 fl/fl Tie2-Cre + mice. Thin sections were stained with hematoxylin and eosin (H&E) (A to D) or examined by immunohistochemistry with anti-Ly-6G antibodies (neutrophils) (E to H) or anti-F4/80 antibodies (macrophages) (I to L). (M to P) Magnifications of boxed area from panels I to L. Macrophages and neutrophils stain brown. Green arrows point to replicating S. aureus cells surrounded by a fibrin capsule. Black bars depict a length of 100 mum. Representative images are shown. (Q) Determination of macrophage-infiltrated areas of renal abscesses of infected C57BL/6 CASP3 fl/fl (black circles) or CASP3 fl/fl Tie2-Cre + mice (open circles) by immunohistochemistry with anti-F4/80 antibodies. Macrophage-infiltrated areas were determined by calculating the total and macrophage-free (anti-F4/80-negative) abscess areas. Multiple abscesses ( n = 25 to 35) from a cohort of 3 to 4 animals per group were analyzed. Horizontal blue bars represent mean values in each cohort. (R) Immunoblotting of lysates from bone marrow-derived macrophages (BMDM). Lysates of BMDM derived from female ( symbol) or male ( symbol) C57BL/6 CASP3 fl/fl or CASP3 fl/fl Tie2-Cre + mice were probed with caspase-3 and GAPDH-specific antibo

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 4 Single nucleotide polymorphisms (SNPs) in CASP3 protect human macrophages from S. aureus -derived deoxyadenosine. (A) Caspase-3 protein lollipop plot highlighting amino acid substitutions investigated in this study. Associated SNP identifiers (IDs) are provided in Table S1 . (B) Immunoblotting of lysates from wild-type (WT) U937-derived macrophages (MPhi) and their SLC29A1 -/- , CASP3 -/- , and complemented CASP3 -/- variants (WT and 12 different alleles indicated according to their amino acid substitution in caspase-3) using caspase-3 and GAPDH-specific antibodies (alpha-CASP3 and alpha-GAPDH, respectively). GAPDH was used as a loading control. Numbers to the left of blots indicate the migration of molecular weight markers in kilodaltons. (C) Caspase-3 activity in cell lysates of dAdo-exposed WT MPhi (black bars) and their SLC29A1 -/- (blue bars), CASP3 -/- (red bars), and CASP3 -/- variants complemented with WT (open bars) and various alleles of CASP3 (gray or pink bars). Caspase-3 activity was measured using a colorimetric assay. (D and E) Survival of WT MPhi (black bars) and their SLC29A1 -/- (blue bars), CASP3 -/- (red bars), and CASP3 -/- variants complemented with WT (open bars) and various alleles of CASP3 (gray or pink bars) after treatment with dAdo (D) or after treatment with culture medium (RPMI) that had been conditioned by incubation with either wild-type S. aureus Newman or its adsA mutant in the presence of host DNA, as indicated with + and - signs (E).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 ZEB1-AS1 interacts with AUF1 to serve key roles in BCa. (A) Immunofluorescence analysis of AUF1 protein in UM-UC-3 cells. Scale bars, 10 u m. (B) The interaction between ZEB1-AS1 and AUF1 was confirmed by RNA pulldown assays and western blot analysis. GAPDH served as negative control. (C) RNA immunoprecipitation assays were performed using anti-AUF1 and control IgG antibodies, followed by RT-qPCR to examine the enrichment of ZEB1-AS1 and U6. U6 served as negative control. ** P