MA3-027

antibody from Invitrogen Antibodies

Targeting: CANX CNX, IP90, P90

Provider product page for MA3-027

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [65]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [6]
Immunohistochemistry [2]
Other assay [23]

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Product number: MA3-027 - Provider product page
Provider: Invitrogen Antibodies
Product name: Calnexin Monoclonal Antibody (AF18)
Antibody type: Monoclonal
Antigen: Other
Description: MA3-027 detects calnexin from human and mouse tissues. MA3-027 has been successfully used in Western blot, immunocytochemistry, immunofluorescence and immunoprecipitation protocols. By Western blot, this antibody detects calnexin in human liver, skin, adrenal gland, heart, colon, testes and ovary extracts. This antibody is not recommended for human kidney or mouse liver lysates in Western blot applications. The predicted molecular weight of this antibody is ~67kDa; however, it may run on SDS-PAGE as an ~90kDA protein. This is due mainly to negative charges at the C-terminus of the protein, which effects the SDS binding to the molecules and consequently its SDS-PAGE mobility. Immunocytochemical staining of calnexin in Huh-7 cells with MA3-027 yields a pattern consistent with specific endoplasmic reticulum staining. The MA3-027 antigen is Focus human hepatoma cell lysate.
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: AF18
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

CANX protein structure - MA3-027 shown in red.

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Experimental details: Immunocytochemical analysis of COS7 cells stained with Calnexin Monoclonal Antibody (Product # MA3-027). Transfected COS7 cells were blocked in 1% BSA, 5% normal goat serum, and 0.1% Triton X-100 in 1X PBS, and then stained with MA3-027 (1:500), followed by a fluorophore-conjugated goat anti-mouse IgG secondary antibody (red). Magnification = 100X. Data courtesy of the Innovators Program.

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Experimental details: Immunofluorescence staining of Calnexin in HMVEC Cells using Product # MA3-027.

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Experimental details: Immunofluorescence staining of Calnexin in A549 Cells using Product # MA3-027.

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Experimental details: Immunofluorescent analysis of calnexin (red) in U87 cells. Cell fixed with 4% paraformaldehyde were permeabilized with 0.05% Triton X-100 in PBS for 60 seconds at room temperature and blocked with 5% normal donkey serum for 30 minutes at room temperature. Cells were probed with a calnexin monoclonal antibody (Product # MA3-027) at a dilution of 1:100 for 2 hours at room temperature, washed with PBS, and incubated with a fluorescently-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:100 for 45 minutes at room temperature. Nuclei (blue) were stained with DAPI. Images were taken on a fluorescent microscope at 40X magnification. Data courtesy of the Innovators Program.

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Experimental details: Immunocytochemical analysis of COS7 cells stained with Calnexin Monoclonal Antibody (Product # MA3-027). Transfected COS7 cells were blocked in 1% BSA, 5% normal goat serum, and 0.1% Triton X-100 in 1X PBS, and then stained with MA3-027 (1:500), followed by a fluorophore-conjugated goat anti-mouse IgG secondary antibody (red). Magnification = 100X. Data courtesy of the Innovators Program.

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Experimental details: Immunofluorescence analysis of Calnexin (red) in MCF7 cells was performed by fixing cells in 4% PFA for 15 min in room temperature. Cells were stained with a Calnexin mouse monoclonal antibody (Product # MA3-027) with a dilution of 1:100 and an incubation overnight at 4C. Confocal imaging was performed using 60X magnification. Data courtesy of Dr Wei Xu's lab at University of Wisconsin Madison.

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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human hepatocarcinoma tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Anti-Calnexin (Product # MA3-027) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human kidney tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:50 with a mouse monoclonal antibody recognizing Anti-Calnexin (Product # MA3-027) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunoprecipitation of CANX was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 ug whole cell lysate with 5 ug of monoclonal CANX antibody (Product # MA3-027) rotating 60 min at RT. The immune complexes were captured on 625 ug of Dynabeads M-280 sheep anti-mouse IgG (Product # 11202D), washed extensively, and eluted in NuPAGE LDS sample buffer (Product # NP0007). Samples were resolved onto a 4-12% Bis-Tris protein gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to nitrocellulose membrane (Product # IB23001) and blocked in 5% milk. CANX was detected using a monoclonal CANX antibody (Product # MA3-027) at a dilution of 1:2000, followed by incubation with anti-mouse secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

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Experimental details: Figure 1. NPGPx expression was induced by non-targeting shRNA/RNAi . ( a and b ) Western blot analysis of NPGPx, p84 and alpha-tubulin proteins from WI38 cells infected with lentivirus carrying shLuc, shRFP, shLacZ, GFP, shEmpty (shRNA cloning vector) or uninfected as control. Specific antibodies against each protein were used as probes. p84 and alpha-tubulin served as internal loading controls. ( c ) Western blot analysis of NPGPx protein level in WI38 cells transfected with various amount of non-targeting siRNA (CTRL siRNA). Relative NPGPx/p84 ratio was shown in below. ( d ) NPGPx mRNA expression analyzed by RT-qPCR from WI38 cells infected with lentivirus carrying with shLuc, GFP or control vector (shEmpty) or uninfected as control (mock). Relative NPGPx mRNA level (normalized with beta-actin and compare with Mock) was shown. ( e and f ) RT-qPCR analysis. WI38 cells transfected with non-targeting siRNA (Ctrl siRNA, 160 nM) or PPIB-1 siRNA were used in this assay. The PPIB-1 mRNA amount (e) and NPGPx mRNA expression level (f) were shown. Mock: WI38 cells without siRNA transfection. ( g ) Western blot analysis of WI38 cells infected with vector control (shEmpty) or shLuc, and then transfected with either pBSK or Luciferase expression vector (pGL3-Luc). Cell lysates were harvested 48 h after transfection for western blot analysis. Relative expression ratio of NPGPx/alpha-tubulin (Rel. NP/alpha-tu) was calculated. ( h ) Expressions of stress-related proteins including NPGPx, C

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Experimental details: RNA immunoprecipitation (RIP) western of CANX was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of monoclonal CANX antibody (Product # MA3-027) rotating 60 min at RT. The immune complexes were captured on 625 µg of Dynabeads M-280 sheep anti-mouse IgG (Product # 11202D), washed extensively, and eluted in NuPAGE LDS sample buffer (Product # NP0007). Samples were resolved onto a 4-12% Bis-Tris protein gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to nitrocellulose membrane (Product # IB23001) and blocked in 5% milk. CANX was detected using a monoclonal CANX antibody (Product # MA3-027) at a dilution of 1:2000, followed by incubation with anti-mouse secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

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Experimental details: Figure 6 The cellular localization of SdeA is not affected by its self-ubiquitination activity. (A) COS-1 cells were transfected to express Flag-SdeA or Flag-SdeA E/A for 24 h. Permeabilized fixed cells were sequentially immunostained with antibodies specific for the ER resident protein calnexin and SdeA; secondary antibodies conjugated to fluorescein and Texas red were used to label calnexin and SdeA, respectively. Note that in both cases, the red fluorescence signals indicative of SdeA co-localized well with those of calnexin. Images were acquired with an Olympus IX-81 fluorescence microscope and were pseudocolored with the IPlab software. Bar, 5 mum. (B) Cellular localization of SdeA or SdeA E/A during bacterial infection. Macrophages infected with L. pneumophila strains expressing SdeA or the SdeA E/A mutant for 2 h were used to determine the cellular localization of translocated SdeA by biochemical fractionation. The ER resident protein calnexin and the cytosolic protein tubulin were used as references. Note that SdeA co-fractionated with calnexin regardless of its ubiquitin ligase activity. Similar results were obtained from three independent experiments. (C) SidJ does not affect the cellular localization of SdeA during bacterial infection. Macrophages infected with wild-type L. pneumophila or a mutant lacking sidJ and sdjA for 2 h were used to determine SdeA distribution in cells by fractionation with sucrose gradient centrifugation. Note that in both samples, SdeA co-

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Experimental details: Fig. 5 Mitochondrial/ER ultrastructure and bioenergetics activity in skeletal muscles. a , b Electron microscopy representative images of control and SOD1 G93A quadriceps ( n = 3). In both panels, red arrows show mitochondria while yellow arrows show ER ultrastructural profiles. c - e Western blot analysis and relative densitometry quantitative analyses of Mfn2, Drp1, and Calnexin in the two groups. f , g ATP synthesis and oxygen consumption rate stimulated by pyruvate/malate in control (green column) and SOD1 G93A (red column) of isolated mitochondria. h Complex I activity in isolated mitochondria form control (green column) and SOD1 G93A muscles (red column) studied by the FeCN reduction in the presence of NADH. Data are expressed as mean +- SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation

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Experimental details: Fig. 1 Generation of PANC-1 cells stably expressing ER-resident and secreted AGR2. a The primary structure of the AGR2 protein shows identified functional domains and motifs. b Expression levels of AGR2 mRNA in PANC-1 stable cell lines were detected by RT-PCR. c Western blotting was used to analyze the presentation of the AGR2 protein in whole cell lysates of PANC-1 stable cell lines. GAPDH served as a loading control. d The presence of AGR2 proteins in cell lysates and culture supernatant was detected by Western blotting. e DSP-stabilized AGR2 in cell lysates and culture supernatant was analyzed under non-reducing (top blot) or reducing conditions (bottom blot). f Cellular distribution of AGR2 and ER-associated protein, calnexin, were determined by immunofluorescence. AGR2 is shown in red, calnexin in green, and Hoechst 33342 in blue. Scale bars: 20 mum

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Experimental details: Figure 5 CCDC170 promoted cell apoptosis under ER stress. ( A ) IF showed that protein localization of CCDC70 overlapped Calnexin partially. Scale bar: 50mum. ( B, D ) Representative western blot bands of Cleaved PARP, Caspase7, Bcl-2 in MCF7 cells when CCDC170 up-regulated transiently ( C ) and stably ( E ) under TG treatment. pCMV-CCDC170(control) represented CCDC170-transiently-overexpressing MCF7 cells and controls. v-CCDC170(control) represented CCDC170-stably-overexpressing MCF7 cells and controls. beta -actin was used as a reference for calculating the relative protein expression. TG: Thapsigargin (300 nm). ( F ) Representative images of flow cytometry using Annexin V-FITC and PI staining, ( G ) Column bar graph showing an increased proportion of early and late apoptotic cells after CCDC170 overexpression in MCF7 cells treated with TG. 3000 cells were calculated. ( H ) Representative images of flow cytometry using Annexin V-FITC and PI staining, ( I ) Column bar graph showing a decreased proportion of early and late apoptotic cells after IRE1 knockdown in MCF7 cells with CCDC170 overexpression. 3000 cells were calculated. CCDC170 OE : MCF7 cells that transiently overexpressed CCDC170. Detection of cell viability via MTT assay. Transient ( J ) or stable ( K ) overexpression CCDC170, the growth of the cell was suppressed in MCF7 breast cancer cells. ( L ) The cell viability of CCDC170-stably-overexpressing MCF7 cells was significantly lower than that of control cells tre

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Experimental details: Figure 3 Subcellular localization of GFP-tagged wild-type and variant PTCHD1 proteins overexpressed in HEK293T cells. (a) Representative confocal microscopy images of HEK293T cells transfected with GFP, PTCHD1 expression plasmids, GFP-tagged wild-type (WT) or variants of PTCHD1, and stained with anti-GFP antibody (PTCHD1-GFP, green), anti-Na + /K + ATPase antibody (plasma membrane, red), and DAPI (nucleus, blue). n= 3 independent transfections. (b) Manders' coefficient of colocalization calculating the percentage of PTCHD1-GFP WT or variants overlapping Na + /K + ATPase (plasma membrane) staining. Kruskal-Wallis test with Dunn's multiple comparisons tests was made to compare each PTCHD1 variant to the WT. Normality of each condition was measured by the d'Agostino-Pearson normality test. For each condition, n = 7-10 images (each one indicated by one respective dot), from three independent transfections. (c) Representative confocal microscopy images of HEK293T cell lines transfected with GFP, PTCHD1 expression plasmids, GFP-tagged wild-type (WT) or variants of PTCHD1, and stained with anti-GFP antibody (PTCHD1-GFP, green), anti-calnexin antibody (endoplasmic reticulum (ER), red) and DAPI (nucleus, blue). n = 3 independent transfections. (d) Manders' coefficient of colocalization calculating the percentage of Ptchd1-GFP WT or variants overlapping calnexin (ER) staining. For each condition, n = 7-10 images (indicated by one respective dot), from three independent transfections. S

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Experimental details: Figure 1 Expression of AQP2-G215S was decreased in the cell membrane compared with AQP2-WT. MDCK cells were transfected with AQP2-WT and AQP2-G215S plasmids. Total membrane proteins (labeled as T) and cytoplasmic membrane proteins (labeled as C) were harvested for AQP2, Pan-cadherin, and calnexin immunoblotting. Pan-Cadherin is the membrane marker, and calnexin is the endoplasmic marker. Protein fold expression normalized to cadherin is shown. WT, wild type; FK, forskolin. Data were shown as mean +- SEM. n = 3, ** p < 0.01, *** p < 0.001, n.s.=no statistically significant difference.

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Experimental details: Figure 3 Endogenous c10orf118 localizes in the Golgi apparatus in MCF-7 cells. ( A ) Confocal microscopy images of MCF-7 cells stained for c10orf118 (green) and Golgin-97 (red), a Golgi apparatus marker. ( B ) Confocal microscopy images of MCF-7 cells stained for c10orf118 (green) and calnexin (red), a specific endoplasmic reticulum marker. ( C ) Line-scan of the staining performed in the section marked with a green line in the merged view and relative quantification. Overlap of the two signals is apparent. ( D ) Line-scan of the staining in the section marked with a green line in the merged view and relative quantification. ( E ) Confocal microscopy images of MCF-7 cells nucleofected with 30 nmol of scrambled siRNA and siRNA against c10orf118 for 48 h and stained with antibody against c10orf118 (green) or Golgin-97 (red). White bars, 20 um.

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Experimental details: Western blot analysis of large extracellular vesicles (EVs), small EVs, and total lysates from C2C12 cell lines (10 mug of protein per lane) to detect the expression of Alix, calnexin, HSP70, and RAB5. Beta-Actin was used as a loading control.

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Experimental details: Characterization of EVs released by SW620, CD9-GFP + SW620, and SW480 cells. ( A - E ) EVs were recovered from the conditioned media of SW620, CD9-GFP + SW620, and SW480 cells by differential centrifugation, and the resulting 200,000x g pellets were analyzed by the ZetaView particle analyzer ( A ), immunoblotting ( B , C ), and dSTORM ( D , E ). The concentration and size of EVs derived from the indicated cells are shown ( A ). Note the presence of a common population of small particles ( 5000 EVs per experiment). Note that small and large EVs derived from SW480 cells were also quantified ( Supplementary Figure S2 ). Scale bars, 50 nm.

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Experimental details: Fig 5 Immunofluorescence microscopy of HEK293A cells shows Surf4 accumulates in and around ERESs. (A) Fluorescent signal for endogenous Surf4 (green) was strongest at punctate structures positive for ERES marker Sec23 (red). Note additional Surf4 fluorescence in weblike structures surrounding ERES. (B) HA-Surf4 signal (green) was observed within the ERGIC (ERGIC-53, red). (C) HA-Surf4 (green) showed only low levels of colocalization with cis -Golgi marker, giantin (red). (D) Mutation of proposed COPI recycling motif by replacement of two of three near-carboxy-terminal lysines to alanines (HA-Surf4-AAK, green) increased colocalization with cis -Golgi marker, giantin (red). (E) Newly synthesized HA-Surf4 (green) was found at low levels in the rER (Sec61 marker, red). (F) Surf4 (green) did not colocalize with chaperone, calnexin (red), in the quality control domain. HEK293A cells were transfected with wild-type HA-tagged Surf4 plasmid (B, C, E), or Surf4 KO HEK293A cells were transfected with carboxyl-terminal di-lysine mutation, HA- Surf4 -AAK (D), 18 hr prior to fixation. Bars = 5mum. The cells in each panel are shown 3 times, first with organelle marker, then Surf4 and final panel (with magnified insert) showing overlap. Alexa Fluor secondary antibodies were used for detection. Images were obtained using an LSM 780 (Carl Zeiss) confocal microscope (488 and 561 nm excitation lines; 500-560 and 600-660 nm capture) and Zeiss Axio Imager Z1 with Apotome 2 (single Z stack slice).

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Experimental details: Fig. 4 Antidepressants-mediated effect on cellular protein content. HT-22 cells were treated with antidepressants for 48 and 96 h and densitometry analysis of NF-kappaB ( b ), p16 ( c ), p21 ( d ), p27 ( e ), p53 ( f ), TRF1 ( g ), TRF2 ( h ), calnexin ( i ), NuMa ( j ), cleaved caspase 3 ( k ), Bcl-2 ( l ) was evaluated. Representative Western Blots are presented ( a ). Bars indicate SD, n = 3, *** /^^^ p < 0.001, ** /^^ p < 0.01, * /^ p < 0.05, no indication--no statistical significance (one-way ANOVA and Dunnett's a posteriori test)