Cells were washed with ice-cold Phosphate-Buffered Saline (PBS) and then lysed in Triton X-100-containing lysis buffer. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. Lysates were precleared by centrifugation before use for Western blotting. Equal amounts of protein were loaded for Western blot analysis. All the following antibodies used were obtained from Cell Signaling Technology: anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-Mcl-1, anti-phospho-Bad (Ser112), anti-Bad, anti-phospho-MEK1/2, anti-MEK1/2, anti-Grb2, anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-calnexin, anti-phospho-p70 S6K1 (Thr389), anti-p70S6K1, except for anti-p27 (BD Biosciences), anti-HIF-1α (BD Transduction Laoratories), anti-GAPDH (US Biological), anti-Glut1 (Abcam), anti-α-tubulin (Molecular Probes), anti-β-actin (Sigma), anti-FLAG M2 (Sigma), anti-V5 (Serotec) and anti-HA (Roche).
Anti calnexin
Manufactured by BD
Sourced in United States
Anti-calnexin is a laboratory-grade antibody used to detect the presence of the calnexin protein in cellular samples. Calnexin is a chaperone protein involved in the proper folding and processing of proteins within the endoplasmic reticulum. The anti-calnexin antibody can be utilized in various analytical techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to identify and quantify the expression levels of calnexin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti α tubulin, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by US Biological (1 mentions)
Anti mcl 1, by BD (1 mentions)
Anti grb2, by BD (1 mentions)
Anti v5, by Bio-Rad (1 mentions)
Anti p27, by BD (1 mentions)
Anti ha, by Roche (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti phospho erk1 2, by BD (1 mentions)
Anti erk1 2, by BD (1 mentions)
Anti bad, by BD (1 mentions)
Anti phospho akt, by BD (1 mentions)
Anti akt, by BD (1 mentions)
Anti phospho stat3, by BD (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti glut1, by Abcam (1 mentions)
Anti stat3, by BD (1 mentions)
10 protocols using anti calnexin
1
Comprehensive Protein Extraction and Analysis
2
Western Blot Analysis of Cell Membrane Microdomains
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Cell membrane microdomains were confirmed using a Western blot analysis. A total of 150 µL of each fraction was precipitated using the methanol–isopropanol–water method [24 (link)], and the protein pellets were dissolved in 50 µL of 1x NuPAGE™ LDS Sample Buffer (Invitrogen, Carlsbad, CA, USA). A total of 20 µL of the protein solution was separated using SDS-PAGE, and gels were transferred to a PVDF membrane (Biorad, Hercules, CA, USA). The PVDF membrane was blocked via incubation with 5% nonfat powdered milk. The PVDF strips were incubated for 1 h (or overnight at 4 °C) at room temperature with a primary antibody (anti-flotillin-1 (Cell Signaling Technology Inc., Danvers, MA, USA) (1:1000), anti-Prohibitins PBH1 (Cell Signaling Technology Inc. Danvers, MA, USA) (1:5000) or anti-Calnexin (BD Biosciences, San Jose, CA, USA)(1:250)); washed; and then incubated with the horseradish peroxidase conjugated secondary antibody (Goat Anti-Rabbit IgG (H + L)-HRP, (Biorad, Hercules, CA, USA), dilution (1:3000) or Goat Anti-Mouse IgG (H + L)-HRP, (Biorad, Hercules, CA, USA), dilution (1:2000)). After washing, antibodies were detected using chemiluminescence with the West Pico PLUS Chemiluminescent-Substrat (Thermo Fischer Scientific, Waltham, MA, USA).
3
Subcellular Localization of SREBP-1c and CREB3L3
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HEK293 cells were transfected with mCherry-tagged pSREBP-1c, SCAP, and GFP-tagged pCREB3L3 using X-tremeGENE 9 (Roche). Cells were grown on coverslips, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 5 minutes. After blocking in 1% bovine serum albumin for 30 minutes, the cells were incubated with primary and secondary antibodies for 1 hour each. The ER and Golgi apparatus were stained using anti-calnexin (610523; BD Biosciences, San Jose, CA) and anti-GM130 antibodies (610822; BD Biosciences), respectively. Immunoreactive complexes were visualized with Alexa Fluor 405-conjugated secondary antibody (ab175660; Abcam, Cambridge, UK), and nuclei were visualized by staining with DAPI.
4
Characterization of Extracellular Vesicles by Western Blot and TEM
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For Western blot analysis (WB), MSC proteins and exosomes were added to 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (PVDF membranes, Millipore), followed by blocking in 5% skim milk for 2 h at RT. Next, the membrane was incubated with diluted primary antibodies (anti-CD9 (BD Biosciences), anti-CD63 (BD Biosciences), anti-TSG101 (BD Biosciences), and anti-calnexin (BD Biosciences)) at 4°C on a shaker overnight. After three washes with Tris-buffered saline/Tween (TBST), the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Proteintech) at RT on a shaker for 2 h. Finally, the membranes were analyzed via the ChemiDoc™ XRS system (Bio-Rad).27 (link)Transmission electron microscopy (TEM, Hitachi, Japan) was used to observe the morphology of the exosomes and take images. Nanoparticle tracking analysis (NTA) was performed by electrophoresis and Brownian motion video analysis laser scattering microscopy (Particlemetrix) to analyze the particle size, concentration and distribution, and the results were analyzed with ZetaView (ZetaView 8.04.02 software).
5
Quantifying SARS-CoV-2 PLpro Activity
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HEK293T cells in 12-well Cell-Bind plate (Corning) were transfected (LT1, Mirus) with 300 ng FLAG-Ub and either 125 ng, 250 ng, or 500 ng of pcDNA3.1-SARS-PLpro per well. Cells were incubated for 18 hours then lysed with IkBα lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 2.5 mM Na pyro-phosphate, 1 mM Beta-glycerophosphate, 1 mM Na ortho-vanadate, 1 ug/ml Leupeptin) and incubated for 20 min on ice. Lysates were subjected to centrifugation at 4C and the cytoplasmic contents added to 2× sample buffer and separated by SDS-PAGE on a 4–20% gradient gel (BioRad). Gel was transferred to PVDF using semi-dry apparatus (BioRad) and immunoblotted with anti-FLAG (Sigma), anti-V5 (Invitrogen), and anti-calnexin (BD).
6
Protein Expression and Immunoblotting Analysis
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Cells were lysed in radioimmunoprecipitation assay buffer and after gel electrophoresis and blotting, membranes were probed with anti-HA (Covance Laboratories, Princeton, NJ), anti-DUOX2,31 (link) anti-Myc (9E10), α-NOXA1,31 (link) anti-NOX1,32 (link) anti-p22phox FL-195 (Santa Cruz Biotechnology, Dallas, TX), anti-calnexin (BD Biosciences, San Jose, CA), and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (SouthernBiotech, Birmingham, AL). Proteins were visualized using enhanced chemiluminescence reagent (Pierce Biotechnology, Rockford, IL). Immunoblotting of p22phox or calnexin served as control.
7
Comprehensive Protein Expression Analysis
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The primary antibodies used were anti-CFTR (596,570) (CFF), anti-TMEM16A (SP31), anti-SLC26A4, anti-GAPDH, anti-βTubulinIV (Abcam, Cambridge, UK), anti-DNAI1, anti-FOXI1 (Sigma-Aldrich), anti-CC16 (BioVendor, Brno, Czech Republic) and anti-Calnexin (BD Biosciences, San Jose, CA, USA).
8
Western Blot Analysis of LPCAT2
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Protein samples were resolved on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (GE Healthcare, Tokyo, Japan) using a Trans-Blot transfer cell (Bio-Rad). Membranes were blocked for more than 16 h with 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA). Anti-LPCAT2 (1:1000), anti-phospho-LPCAT2 (1:1000), and anti-calnexin (1:100) (BD Biosciences) antibodies were used as the primary antibodies. Anti-LPCAT2 and anti-phospho-LPCAT2 antibodies were available from another study (13 (link)). Horseradish peroxidase–conjugated secondary antibodies (1:2000; GE Healthcare) were used. ECL select Western blot detection system (GE Healthcare) was used for chemiluminescence, and detected using ImageQuant LAS500 (GE Healthcare).
9
Immunofluorescence Assay for Organelle Localization
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Cells were fixed in 4% paraformaldehyde for 10 min and permeabilized for 30 min in 0.15% Triton-X100 in PBS + 3% BSA. Incubation with primary antibodies was performed for 2 hrs in 0.15% Triton-X100 in PBS + 3% BSA at room temperature. dsRNA, EGFR, and EEA1 immunolocalization was performed using anti-dsRNA J2 (Scicons) [76 (link)], anti-EEA1, anti-Calnexin, anti-puromycin (BD Biosciences), anti-LARP1 (Novus Biologicals), anti-NS5A 9E10 (gift from C.Rice), and anti-EGFR (Millipore) antibodies (2 μg/mL). For puromycylation assays, 200 μM emetin (Sigma) and 90 μM puromycin (Sigma) were incubated on cells for 10 min before harvest. Samples were incubated with Alexa-488-goat anti-mouse and Alexa-594-goat anti-rabbit antibodies (Invitrogen, 1 μg/mL) for 1 h at room temperature. Nuclei were counterstained with Hoechst 33342. Images were acquired using a Leica SP5X confocal microscope equipped with LAS AF software. Subsequent analyses were performed using the JACop ImageJ colocalization plugin (http://rsb.info.nih.gov/ij/plugins/track/jacop.html ) and its plot profile function.
10
CFTR Protein Expression Analysis
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Cells were lysed using a lysis buffer (31.25 mM Tris-HCl pH 6.8, 1.5% SDS [w/v], 5% glycerol and 0.5 mM DTT) supplemented with complete protease inhibitor cocktail (Roche, Basel, Switzerland) and Laemmli sample buffer (#1610747, Bio-Rad). Whole-cell lysates were then subjected to SDS-PAGE 10% gel (w/v) and transferred to a PVDF membrane (Millipore, MA, USA). CFTR was detected using the monoclonal anti-human CFTR antibody 596 (1:3000, from CF Foundation Therapeutics [CFFT]) and the blotting-grade horseradish peroxidase secondary antibody (1:3000, Bio-Rad). Anti-calnexin (1:3000; BD Biosciences) or anti-tubulin antibodies (1:10,000; Sigma-Aldrich) were used as a loading control. Proteins were detected using the antibodies mentioned above and subsequently visualized by chemiluminescence using the Clarity Western ECL substrate (Bio-Rad) in a Chemidoc XRS system. Bands were analyzed with Image™ Lab software version 6.0 (Bio-Rad).
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