13-8800

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of BCL-2 was performed by loading 20 µg of Raji (lane1), MCF7 (lane2), Jurkat (lane3), A549 (lane4) and HEK-293 (lane5) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. BCL-2 was detected at ~27 kDa using BCL-2 Mouse Monoclonal Antibody (Product # 13-8800) at 1-3 µg/mL dilution in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Mouse IgG - HRP Secondary Antibody (Product # G-21234) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Bcl-2 expression in Jurkat cells using Ms x BCl-2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of BCL-2 was performed by loading 20 µg of Raji (lane1), MCF7 (lane2), Jurkat (lane3), A549 (lane4) and HEK-293 (lane5) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. BCL-2 was detected at ~27 kDa using BCL-2 Mouse Monoclonal Antibody (Product # 13-8800) at 1-3 µg/mL dilution in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Mouse IgG - HRP Secondary Antibody (Product # G-21234) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of Bcl-2 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID ) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Bcl-2 was performed by loading 30 µg of HeLa Cas9 (Lane 1) and andHeLa Bcl-2 KO (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Bcl-2 Monoclonal Antibody (Bcl-2-100) (Product # 13-8800, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Bcl-2. An uncharacterized band was observed at ~38 kDa in both the samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Bcl2 was performed by loading 2 µL SeeBlue® Plus2 Prestained Protein Ladder (Product # LC5925) (lane 1) and 50 µg HeLa (lane 2, HeLa (human) cell lysate onto a 10% Tris-Glycine polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 1% BSA/TBST for at least 1 hour at room temperature. Bcl2 was detected using a monoclonal antibody (Product # 13-8800) at a concentration of 1 µg/mL in blocking buffer overnight at 4°C on a rocking platform, followed by a goat anti-mouse IgG Alexa Fluor 680 secondary antibody (Product # A11357) at a dilution of 1:10,000 for at least 1 hour. Fluorescent detection was performed using the Odyssey® CLx imaging system (Li-cor Biosciences). Images generated by Joell Solan in Paul Lampe Lab at Fred Hutchinson Cancer Research Center.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Immunohistochemistry for p16 ( A ) and bcl-2 ( B ), showing diffuse strong reactivity (x40).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Effect of Oleanolic acid (OA) on proteins involved on apoptotic and metastatic pathways. (Upper panel) Immunofluorescence of A549 cells stained for Bcl2 (A-B), Bax (C-D), survivin (E-F) and VEGF (G-H). A549 cells were treated with medium (A,C,E,G) or with 50 ug/mL OA (B, D, F, H) for 24 h, fixed, stained with the primary antibodies, revealed with biotinilated IgG followed by streptavidin-Cy3 and counter stained with DAPI, as described in M&M. Representative immunofluorescence micrographs of three experiments. Bar: 100 um. (Lower panel) Graphical representation of the histomorphometrical results of the immunocytochemical assay. The percentage of immunoreactive cells was calculated from the DAPI positive cells. Bcl-2 was not modulated by OA (p>0.05), but Bax was significantly increased (* p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Western blot analysis. A549 cells were treated with medium or with 50 ug/mL Oleanolic acid for 24 h and whole cell extracts were obtained as described in M&M. Proteins were separated by SDS-PAGE, transferred to PDF membranes, probed with antibodies to Bcl-2, Bax, Survivin, VEGF or alpha-tubulin and developed with ECL, as described in M&M. Numbers represent band intensity in relation to alpha-tubulin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 In vitro silencing activity of anti- BCL2 U1 Adaptors and a tumor-targeting nanoparticle . ( a ) Design and sequences of the BCL2-A and BCL2-B Adaptors, backbone-modified variants of BCL2-A and matching controls as well as an anti- BCL2 DsiRNA used as a positive control. The boxed region is to indicate a full set of matching control Adaptors. ( b ) The BCL2-A L2 U1 Adaptor reduces BCL2 protein. At 24 hours post-transfection of the indicated Lipofectamine 2000 (LF2000):Adaptor or LF2000:anti- BCL2 DsiRNA complexes, total C8161 lysates were prepared followed by western blot. Lanes 3-10 contain 50 ug/well while lane 2 has 17 ug of protein to show the dose response of the western blot signal. Tubulin was used to show equal loading (top panel) and the lower panel was probed for BCL2. Quantitation of the western signals is shown as a BCL2:tubulin ratio with lane 3 set to 1.0. ( c ) Schematic of RGD-G5 (MW ~9,600) that has a 2:1 RGD to PPIG5 conjugation ratio. For simplicity, only some of the 64 free amino groups of PPIG5 are shown. The attachment positions of the SM(PEG) 12 -linker-RGD group are random and are shown here for illustrative purposes. ( d ) The RGD-G5 nanoparticle (right panel) is comparable to LF2000 (left panel) at delivering the BCL2-A Adaptor to C8161 cells as measured by RT-qPCR of BCL2 mRNA normalized to HPRT1 . Note Cq values between the LF2000 control and RGD-G5 control were comparable. DsiRNA, Dicer-substrate small interfering RNA; LNA, locked nucleic

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 BCL2-A suppresses tumor growth in vivo . ( a ) The design and sequences of the BCL2-A and matching control U1 Adaptors are shown at the top. C8161 xenograft mice were treated when tumor volumes reached ~10 mm 3 , which is denoted as day 1. Each treatment group (TG) mouse was treated two times/week consisting of a tail vein injection of 50 ul 1x PBS containing BCL2-A or matching control U1 Adaptors in complex with the RGD-G5 vehicle or the G5 vehicle that lacked RGD (TG6). The only TG that showed significant tumor suppressive activity was TG2 in comparison with TG1. Note that 1.7 ug Adaptor is ~0.16 nmol, 2.4 ug RGD-G5 is 0.2 nmol. ( b ) Immunoprecipitation (IP)-western blot of day 22 excised tumor samples. Due to the low BCL2 signal in standard western blots using excised tumor lysates in comparison to cultured cells (compare lane 9 with lanes 7-8, 10-11), we performed anti-BCL2 IP in lanes 3-6 before the western blot. Antitubulin IP was also done in lanes 3-6 to internally control for IP efficiency. Lane 2 was antitubulin IP only to show the specificity of the BCL2 IP signals. Lane 1 had 3x less protein than lane 3 to show the dose response of the IP-western blot signals. Levels of BCL2 protein were comparable in TG1, TG3, and TG4 (lanes 3, 5, and 6) whereas TG2 (lane 4) BCL2 levels were barely detectable. Quantitation of the western signals is shown in lanes 3-6 as a BCL2:tubulin ratio with lane 3 set to 1.0. ( c , d ) Immunohistochemistry of day 22 excised tumor s

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Various intensity stain of odontogenic epithelium in dental follicle with bcl-2 (x400). int=1 (a), int=2 (b), int=3 (c)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Case #1. Strong bcl-2 and weak EMA expression by neoplastic cells ( Left : Immunohistochemistry, anti-bcl-2 Ab x 200; Right : Immunohistochemistry, anti-EMA Ab x 200)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 FADD augments downstream apoptosis signaling in TNF-alpha stimulated cells. TNF-alpha (10 ng/ml) was subjected to HEK 293T cells and 48 h of pcDNA-FADD transfected HEK 293T cells. Control represents HEK 293T cells without TNF-alpha treatment (black bar) and 48 h pcDNA-FADD transfcted HEK 293T cells (white bar) (shown as 0 h time point). ( a ) Percent cell viability, ( b ) Percent cell proliferation, ( c ) Images of Propidium iodide (PI) staining (pEYFP-FADD construct was used in this experiment) (scale bar-2 mum), ( d ) Apoptotic cell death monitored by Flow cytometric analysis using (BD FACSAria 3, BD Biosciences, San Jose, CA, USA BD), the result represents in contour plots with quadrant gates showing early apoptotic shown in quadrant 4 (Q4) and late apoptotic in quadrant 2 (Q2), ( e ) Percent apoptotic death (Annexin-V-FITC + /PI + ) by Tali TM image based cytometer, ( f) Expression of of cell death regulatory proteins examined by Western blotting, (g) caspase-8 activity, ( h ) caspase-3 activity. The uncropped full-length blot of Procaspase-8, processed caspase-3 and cytochrome c are presented in supplementary Fig. S10 . Next, HEK 293T cells were treated as mentioned in Figure legend 2i, TNF-alpha untreated and non targeting siRNA transfected cells were taken as control. Here results illustrate ( i) Quantitative analysis of loss of mitochondrial membrane potential (Psi), ( j ) Percent apoptotic death (Annexin-V-FITC + /PI + ) by Tali TM image based cytometer, ( k

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Time course of transient Bcl-2 expression in NSCs. (a) Flow cytometric quantification of Bcl-2 positive cells over 1 week posttransfection with minicircle constructs (circle) or posttransduction with adenovirus (square). (b-c) Representative immunofluorescent images demonstrating a decline in eGFP and Bcl-2 expression in both minicircle-transfected (b) and adenovirally transduced NSCs (c) over 1 week. Scale bars in both (b) and (c) = 50 mu m.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 The immunotoxin hD7-1(VL-VH)-PE40 induces apoptosis in PSMA-positive cells by reducing the expression of Mcl-1 and Bcl2A1. PC cells were incubated for different time points with 1 nM immunotoxin. After cell lysis, expression of the anti-apoptotic Bcl-2 proteins Bcl-2, Bcl-xl, Mcl-1, Bcl-w, and Bcl2A1, the pro-apoptotic Bcl-2 proteins Bax and Bak, as well as the activation of caspase-3, and -8, and the cleavage of PARP were determined by Western blot. ss-actin was detected as loading control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Effects of GH and IGF-1 treatments upon activation of the PI3K/Akt and MAPK/ERK1/2 signaling pathways and Bcl-2 expression after a hypoxic-ischemic injury in primary cerebellar cultures. Cells were maintained under either normoxic conditions (Nx), submitted to hypoxic-ischemic injury for 12 h (HI) and then reoxygenated for 24 h (HI + Ox), or treated with 1 nM GH or 40 nM IGF-1 after (denoted by A) 12h of HI, along the 24 h of reoxygenation period (HI + Ox GH-A; HI + Ox IGF-1-A). The upper panels show Western blots with specific immunoreactive bands corresponding to the following proteins: ( A ) total Akt (60 kDa) or pAkt (S473, 60 kDa); ( C ), ERK1/2 (44 kDa) or pERK1/2 (T202/Y204, 44 kDa); ( E ), Bcl-2 (27 kDa); and GAPDH (panels ( A , C , E ), 37 kDa) as loading control. ( B , D , F ) show densitometric (% O.D.) analysis of changes observed in ( A , C , E ). Bars represent the mean +- SEM, n = 5 independent experiments performed in duplicate. Groups with different letters are significantly different by one-way ANOVA and Tukey's post hoc test ( p < 0.01).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 BAX/BCL-2 ratio had no significant changes in the presence of TPGS in K562 cells. K562 cells were incubated with increasing concentrations of TPGS (10-80 mu M) at 37degC for 24 h. (a) Protein extracts were blotted with primary antibodies as listed. (b-d) Relative infrared fluorescence of (b) BCL-2, and (c) BAX antibodies were expressed as fold increase relative to that in untreated cells and normalized to actin. (d) BAX/BCL-2 ratio. Data are expressed as the mean +- SD; * p < 0.05, ** p < 0.005, and *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. miR-205-3p antagomir inhibits lung cancer growth and promotes apoptosis in mouse xenografts. (A) Representative images of excised xenograft tumors and (B) tumor volumes from mice treated with the antagomir NC or miR-205-3p antagomir. (C) Reverse transcription-quantitative PCR analysis was used to detect miR-205-3p expression in tumors. (D) Western blot analysis was used to detect APBB2, Bax and Bcl-2 protein expression levels in tumors. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 miR-15 and miR-16 induce the apoptosis and fibrosis of pancreatic stellate cells by targeting Bcl-2 and Smad5. a miR-15b and miR-16 share conserved binding sites in 3'UTR of Bcl-2 and SMAD5. b The luciferase activity of either wildtype (Wt) or mutant (Mut) 3'UTR of Bcl-2 and SMAD5 in PSCs after overexpression of miR-15 or miR-16. c The protein levels and the related quantification of Bcl-2 and SMAD5 in PSCs after overexpression of miR-15 or miR-16. d The protein levels and the related quantification of Bcl-2 and SMAD5 in pancreatic tissues of rats with or without chronic pancreatitis after treated with Saline or SAHA (25 mg/kg). All data are presented as the mean +- S.D. ( n = 3). *** P < 0.001, compared with control. Each assay was performed in triplicate

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. miR-125a-5p inhibits proliferation and promotes apoptosis via tafazzin phospholipid-lysophospholipid transacylases signaling. Cellular inhibition rates were determined using CCK-8 assays. MCF-7/Adr cells were treated with various concentrations of Adr or Doc for 72 h. Inhibitory effects were increased with miR-125a-5p transfection. The IC 50 was reduced from 5.99 uM (NC) to 2.28 uM (miR) in the MCF-7/Adr cells (A) treated with Adr, and from 5.01 uM(NC) to 1.63 uM (miR) in the cells (B) treated with Doc. MCF-7/Adr cells were transfected with miR-125a-5p and treated with (C) Adr (2.28 uM) or (D) Doc (1.63 uM) for 24, 48, 72, 96 and 120 h. The CCK-8 assay results indicated that this combination inhibited MCF-7/Adr cell viability. (E and G) Apoptotic rate of MCF-7/Adr breast cancer cells treated with Adr, as detected via flow cytometry. (F and H) Apoptotic rate of MCF-7/Adr breast cancer cells treated with Doc as detected via flow cytometry. a, NC group; b, drug group; c, miR (miR-125a-5p) group; d, miR/drug group. Caspase-3 activation and increased Bax expression were observed in MCF7/Adr cells transfected with miR-125a-5p and treated with (I) Adr or (J) Doc; the combination of miR-125a-5p and Adr or Doc kept Bcl-2 and PCNA expression at the lowest level, as determined via western blot analysis. GAPDH served as an internal control. (K and L) Semi-quantification of protein expression levels in panel (I). (M and N) Semi-quantification of protein expression levels in pane

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7. Effect of miR-183-5p.1 overexpression on the expression of apoptosis-associated proteins in GC AGS cells as detected by western blot analysis. (A and B) P53 was decreased and Bcl-2 was increased in the miR-183-5p.1 mimic-transfected cells. (C and D) miR-183-5p.1 inhibitors significantly inhibited the expression of Bcl-2, but significantly enhanced the expression of p53. GAPDH was used as the internal control. Results are presented as the mean +- standard error of the mean (n=3). ***P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Effect of growth hormone (GH) upon Bcl-2 and TNF R2 immunoreactivity and BDNF, BMP4 and GH mRNAs expression in chicken retinas exposed to excitotoxic damage.( A ) Representative western blot luminograms for Bcl-2 and TNF-R2 immunoreactivities in experimental groups. beta-Actin was included as a loading and normalizing control. 1, 2 and 3 represent different samples. Immunoreactive bands were analyzed by densitometry for TNF-R2 ( B ) and Bcl-2 ( C ). Relative expression of: ( D ) BDNF, ( E ) BMP4, and ( F ) GH mRNAs, as determined by RT-qPCR. Relative mRNA expression values were corrected by the comparative threshold cycle (Ct) method and employing the formula 2 -CT . Ribosomal 18S RNA was used as housekeeping gene. Bars represent mean +- SEM ( n = 4; analyzed by duplicate). Asterisks indicate significant difference in comparison to control (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001) and number sign (#) shows difference between experimental groups (#, p < 0.05; ####, p < 0.0001) as determined by one-way ANOVA for multiple comparisons and Sidak as post-hoc test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. Lack of hTim8a, but not hTim8b, sensitises cells to apoptotic-cell death. ( A ) Mitochondrial lysates from control and hTim8a KO (left panel) or hTim8b KO (right panel) HEK293 cells were analysed by SDS-PAGE and western blotting. Relative protein levels of cytochrome c (Cyt c ), apoptosis-inducing factor (AIF), Bax and Bak were quantified and tabulated as mean +- SD (n = 3). **, p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 miR-181c-5p inhibited cell proliferation and promoted cell apoptosis in SiHa cells. Cells were divided into four groups: control group, untransfected SiHa cells; mimic group, SiHa cells transfected with miR-181c-5p; pc-GSKIP group, cells transfected with overexpression vector of GSKIP; mimic+pc-GSKIP group, cells co-transfected with mimic and pc-GSKIP. ( A ) MTT was used to detect the rate of cell cloning rate. ( B ) Flow cytometry was used to detect the apoptosis rate. ( C ) Western Blot was used to detect levels of proteins associated with proliferation and apoptosis. The experiments were repeated three times and the data are represented as means +- SD; * P