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Anti calnexin

Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom

Anti-calnexin is a laboratory reagent used to detect the presence of the calnexin protein in cellular samples. Calnexin is a chaperone protein involved in the folding and quality control of newly synthesized glycoproteins within the endoplasmic reticulum. The anti-calnexin antibody can be used to identify and quantify calnexin levels in various cell and tissue types.

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54 protocols using anti calnexin

1

Subcellular Protein Extraction and Immunoblotting

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All cells were briefly rinsed with cold PBS prior to collection. Both whole cells and subcellular fractions were lysed in RIPA buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 0.5% SDS, 0.5% sarkosyl, 0.5% NP40, 20 mM EDTA, Roche protease inhibitors, 1 mM PMSF) on ice. Protein concentrations of lysates were determined by the bicinchonic acid assay (Thermo Fisher) before being analyzed by SDS-PAGE. The primary antibodies used included anti-CARM1 (Cell Signaling Technology, 3379), anti-skp2 (Santa Cruz Biotechnology, sc-7164), anti-LC3B (Cell Signaling Technology, 3868), anti-LAMP1 (Cell Signaling Technology, 3243), anti-C9orf72 (Bio-Rad VMA00065), anti-PARP (Cell Signaling Technology, 9542), anti-β-actin (Santa Cruz Biotechnology, sc-47778), anti-NOX2 (Abcam, ab129068), anti-ubiquitin (Cell Signaling Technology, 3936), anti-calnexin (Cell Signaling Technology, 2679P), anti-ACC (Cell Signaling Technology, 3676), and anti-ADRP (Progen Biotechnik, GP42).
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2

Comprehensive Protein Expression Analysis

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The primary antibodies used were anti-collagen I, anti-VEGF and anti-PDGFBB (Abcam, Cambridge, UK); anti-TSG101, anti-CD9, anti-CD63, and anti-CD81 (System Biosciences, Mountain View, CA, USA). The primary antibodies anti-CD41, anti-TGF-β, anti-bFGF, anti-cleaved-caspase-3, anti-Runx2, anti-β-catenin, anti-β-tubulin, anti-calnexin, anti-Bcl-2, anti-CHOP, anti-Bad and phosphorylated Bad (p-Bad), anti-Erk1/2 and phosphorylated Erk1/2 (p-Erk1/2), anti-Akt and phosphorylated Akt (p-Akt) antibody were all obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-PERK and phosphorylated PERK (p-PERK) antibodies were obtained from Santa Cruz Biotechnology.
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3

Immunoblotting Analysis of Protein Expression

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Analysis of protein expression by immunoblotting was performed as previously described (10 (link)). Proteins of cell lysates (20–40 μg) were dissolved in SDS sample buffer, separated by 10% or 12.5% SDS-PAGE, and transferred onto a polyvinylidene fluoride membrane (Carl Roth GmbH, Karlsruhe, Germany). The membrane was blocked with 10% nonfat dry milk and incubated with the following primary antibodies: anti-ATGL, anti-HSL, anti-GAPDH, and anti-calnexin from Cell Signaling Technology (Danvers, MA; 2138S/ATGL, 4107S/HSL, 2118S/GAPDH, 2679S/calnexin), anti-6x HIS tag, and anti-NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) from Abcam (Cambridge, England; ab18184/6x HIS-tag®, ab157221/NDUFS1), anti-α-SMA from Thermo Fisher Scientific (Waltham, MA; PA5-22251/α-SMA), and anti-KIAA1363 from Invitrogen GmbH (PA5-50285/NCEH1 = KIAA1363), respectively. For detection, membranes were incubated with horseradish peroxidase-labeled secondary antibodies specific for respective primary antibody. Bands were visualized using the ECL plus Western blotting Detection Reagent (Thermo Fisher Scientific) and ChemiDoc Touch Imaging System (Bio-Rad).
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4

Antibody Characterization in Mouse Models

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The following primary mouse antibodies were used: anti-actin (Sigma-Aldrich, St. Louis, MO, United States, catalog No. A2228), anti-Flag (Sigma-Aldrich, St. Louis, MO, United States, catalog No. A2220), IgG control (MBL, Nagoya, Japan, catalog No. M075-3), anti-SR-BI (BD Biosciences, Franklin Lakes, NJ, United States, catalog No. 610883), and anti-UGGT1 (Santa Cruz Biotechnology, Santa Cruz, CA, United States, catalog No. Sc-374565). The following primary rabbit antibodies were used: anti-calnexin (Cell Signaling Technology, Boston, MA, United States, catalog No. 2433), anti-SR-BI (Novus Biological, Centennial, CO, United States, catalog No. NB400-104). The secondary antibodies included: HRP-conjugated ECL goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, United States, catalog No. A6154), HRP-conjugated ECL goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, United States, catalog No. A4416), donkey anti-mouse-Alexa Fluor 488, donkey anti-rabbit-Alexa Fluor 594, donkey anti-rabbit-Alexa Fluor 488, donkey anti-mouse-Alexa Fluor 594, donkey anti-rat-Alexa Fluor 488 (Invitrogen, Carlsbad, CA, United States).
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5

Immunodetection of Tight Junction Proteins

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The following primary monoclonal and polyclonal antibodies were used to detect proteins by immunofluorescence (IF) or immunoblot (IB). From rabbit: anti-human/mouse CLDN23 (IB: 1/1,000; IF: 1/100) was generated; anti-human/mouse CLDN3 (Sigma, Cat. 218317, IB:1/1000; IF:1/100); anti-human CDX2 (Cell Signaling, Cat. 39775, IB: 1/1000); and anti-calnexin (Cat. PA5-34665; IB: 1/20,000). From mouse: anti-mouse CLDN2 (Invitrogen, Cat. 32-5600; IB: 1/1000; IF: 1/250); anti-human CLDN3 (Sigma, Cat. SAB4200758, IB:1/1,000; IF:1/100); and anti-human CLDN4 (Invitrogen, Cat. 32-9400, IB:1/2000; IF:1:200); anti-human/mouse ZO-1 (Thermofisher, Cat. 33-9100, IB: 1:1000, IF 1:100).
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6

Antibody Panel for Vesicle Characterization

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For western blotting, immunoprecipitation and immunofluorescence, the following antibodies were used: anti-ANXA2 (Abcam, ab54771, for confocal microscopy; BD, 610069, for western blotting), anti-CD63 (Abcam, ab18235), anti-Tsg101 (Abcam, ab83), mouse IgG (BD, 554121), anti-ATG5 (Cell Signaling Technology, 2630), anti-calnexin (Cell Signaling Technology, 2433), anti-LAMP1 (Cell Signaling Technology, 9091), anti-RAB7 (Cell Signaling Technology, 9367), anti-LC3 (MBL, PM036), anti-RAB8A (Proteintech, 55296-1-AP), anti-RAB27A (Proteintech, 17817-1-AP), anti-RAB27B (Proteintech, 13412-1-AP), anti-VAMP7 (R&D Systems, MAB6117), anti-cathepsin S (Santa Cruz, sc-74429), anti-SQSTM1/p62 (Santa Cruz, sc-28359), and anti-α-tubulin (Santa Cruz, sc-5286). For secondary staining in western blotting, the following antibodies were used: HRP-linked anti-mouse IgG (Cell Signaling Technology, 7076) and HRP-linked anti-rabbit IgG (Cell Signaling Technology, 7074). For secondary staining in immunofluorescence, the following antibodies were used: Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, A11034), Alexa Fluor 594 donkey anti-mouse IgG (Life Technologies, A21203), Alexa Fluor 594 donkey anti-rabbit IgG (Life Technologies, A21207), Alexa Fluor 647 donkey anti-mouse IgG (Life Technologies, A31571), and Alexa Fluor 647 donkey anti-rabbit IgG (Life Technologies, A31573).
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7

Characterization of Extracellular Vesicles

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EV sample was concentrated using Concentrator plus 5305 Vacuum Centrifuge (Eppendorf AG, Germany), and protein concentration was measured with Pierce BCA Protein Assay Kit (Thermo Scientific). Concentrated sEV preparations and lysate of HCT116 cells were lysed in Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific), heated for 5 min at 95 °C, and subjected to electrophoresis using 10% SDS-PAGE. Proteins were transferred to an Immobilon-P PVDF Membrane (Merck Millipore) and the excess protein binding sites on the membrane were saturated with 5% bovine serum albumin blocking buffer (1 × TBS, 0.1% Tween-20) for 1 h. The membrane was incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-CD81 (1:250, mouse, catalogue number sc166029), anti-CD63 (1:300, mouse, sc5275) from Santa Cruz Biotechnology, anti-Alix (1:20000, rabbit, ab186429) from Abcam, anti-TSG101 (1:200, mouse, 612696) from BD Biosciences, and anti-Calnexin (1:1000, rabbit, 2679) from Cell Signaling. After incubation, the membrane was washed three times with 5% TBS-Tween and then, incubated with peroxidase-labelled secondary antibody (Santa Cruz Biotechnology) for one hour. After three washes, immobilized proteins were detected utilizing Clarity Western ECL Substrate (Bio-Rad) and the UVITEC chemiluminescence imager (UVITEC Cambridge, UK).
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8

Ago Protein Immunoprecipitation and Analysis

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THP-1 cells (TIB-202TM) were from ATCC. RPMI 1640 media and Fetal bovine serum (FBS) were purchased from Gibco. HEPES, penicillin/streptomycin, L-glutamine, Hanks’ balanced salt solution, M199 medium, folic acid, hemin, adenosine, Pepstatin A, Phorbol 12-myristate 13-acetate (PMA), PreScission enzyme (GE27-0843-01), and Amicon® Ultra-2 centrifugal filter devices (UFC200324) were purchased from Millipore Sigma. Anti-Ago1 (5053), anti-Ago2 (2897), anti-Ago3 (5054), anti-Lamin A/C (2032), anti-Calreticulin (12238), and anti-Calnexin (2679) were purchased from Cell Signaling. Anti-GST (SC-138), anti-HSC70 (HSP70) (SC-7298), and anti-Actin (SC-47778) were from Santa Cruz biotechnology. Anti-GAPDH (G041) was from Abm, and anti TRBP (Ab42018) was purchased from Abcam. RPMI 1640 media lacking L-glutamine, L-arginine, and L-lysine was purchased from Caisson labs. North2South Hybridization buffer (37549), North2South Hybridization Stringency Wash Buffer (37555), and Chemiluminescent Nucleic Acid Detection Module kit (89880) were purchased from ThermoScientific.
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9

ER Stress-Induced Apoptosis and Autophagy

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TBT and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell lysis buffer, fetal bovine serum (FBS), RPMI 1640 medium, and Western Bright Enhanced Chemiluminescence (ECL) detection reagents were obtained from Thermo Fisher Scientific (Suwanee, GA, USA). An Annexin V/PI apoptosis detection kit was purchased from Multi-Sciences (Hangzhou, China). Anti-Bip (Cat#3177), anti-Calnexin (Cat#2679), anti-Ero1-Lα (Cat#3264), anti-IRE1α (Cat#3294), anti-PDI (Cat#3501), anti-CHOP (Cat#2895), anti-PERK (Cat#5683), anti-eIF2α (Cat#5324), anti-P-eIF2α (Cat#3398), anti-Atg12 (Cat#4180), anti-Beclin-1 (Cat#3495), anti-JNK (Cat#3708), anti-P-JNK (Cat#4668), anti-LC3A/B (Cat#12741), anti-Atg5 (Cat#12994), anti-Atg16L1 (Cat#8089), anti-Atg7 (Cat#8558), anti-Atg3 (Cat#3415), anti-rabbit IgG (Cat#7074), and anti-mouse IgG (Cat#7076) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-ATF6 (Cat#ab227830) was purchased from Abcam (Cambridge, UK) and anti-GAPDH was obtained from KangChen Biotech (Shanghai, China).
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10

Antibody Selection for Western Blotting and Immunoprecipitation

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Antibodies used for western blotting were as follows: rabbit polyclonal anti-Rer1 (R4407, Sigma-Aldrich, St. Louis, MO, USA), anti-Hrd1 (H7915, Sigma), anti-gp78 (9590, Cell Signaling Technology, Danvers, MA, USA), a rabbit monoclonal anti-Calnexin (C5C7, Cell Signaling), goat polyclonal anti-GFP (RDI, Fitzgerald Industry, Concord, MA, USA), mouse monoclonal anti-α-actin (C4, Millipore, Billerica, MA, USA), anti-FLAG (M2, Sigma-Aldrich), and anti-GSK3β (610201, BD Transduction Laboratories, San Diego, CA, USA) antibodies. Antibodies used for immunoprecipitation were as follows: mouse monoclonal anti-GFP (3E6, Q-Biogene, Carlsbad, CA, USA), anti-α-tubulin (DM1A, Sigma-Aldrich), and anti-FLAG (M2, Sigma-Aldrich) antibodies. Antibodies used for immunocytochemistry were as follows: rabbit polyclonal anti-Rab7 (a gift from Y. Wada, Osaka University)49 (link), anti-ERGIC-53 (E1031, Sigma-Aldrich), mouse monoclonal anti-PDI (1D3, Enzo Life Sciences, Plymouth Meeting, PA, USA), and anti-Lamp1 (H4A3, Santa Cruz Biotechnology, CA, USA) antibodies. Alexa Fluor® 555 or 594-conjugated anti-mouse or anti-rabbit immunoglobulin Gs (IgGs) (Life Technologies) were used as secondary antibodies.
MG132 was purchased from the Peptide Institute (Osaka, Japan). Bafilomycin A1 was purchased from Wako Pure Chemicals (Osaka, Japan). Cycloheximide was purchased from MP Biomedicals (Solon, OH, USA).
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