Cells were lysed in RIPA buffer containing complete mini protease inhibitor (Roche) for 15 min on ice with occasional vortexing. Samples were centrifuged, and cell lysates were mixed with SDS loading buffer and boiled at 95°C for 10 min. Sample were then loaded and separated on 10% SDS-PAGE gels, before transferring onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% milk diluted in PBS containing 0.05% Tween-20 (PBST) for 1 hour, before incubation with primary antibodies at 1:500-1000 dilution overnight at 4°C. Thereafter, membranes were washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h before detection with Western Lightning Chemiluminescence (Perkin Elmer). Antibodies used included: anti-EBNA1 (sc-57719, Santa Cruz), anti-EBNA2 (ab90543, Abcam), anti-LMP1 (CS1-4, Dako), anti-LMP2A (MCA2467, Bio-rad), anti-GAPDH (mab374, Merck), anti-mouse IgG-HRP (ThermoFisher Scientific) and anti-rat IgG HRP (Santa Cruz).
Anti gapdh mab374
Manufactured by Merck Group
Sourced in United States
Anti-GAPDH (MAB374) is a monoclonal antibody that specifically recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a key enzyme involved in the glycolysis pathway, which is essential for energy production in cells. This antibody can be used as a tool for the detection and quantification of GAPDH in various applications, such as Western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Kifunensine, by Merck Group (2 mentions)
Pugnac, by Merck Group (2 mentions)
Pd98059, by Cell Signaling Technology (2 mentions)
Anti man1a1 m, by Merck Group (2 mentions)
Anti pfoxo3 thr 32, by Cell Signaling Technology (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Ab90543, by Abcam (1 mentions)
Cs1 4, by Agilent Technologies (1 mentions)
Anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti rat igg hrp, by Santa Cruz Biotechnology (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Anti ubiquitin ab7780, by Abcam (1 mentions)
Anti fip200 17250 1 ap, by Proteintech (1 mentions)
Anti ubiquitin p4d1 sc 8017, by Santa Cruz Biotechnology (1 mentions)
Anti galectin 8 af1305, by R&D Systems (1 mentions)
Anti vimentin, by GeneTex (1 mentions)
31 protocols using anti gapdh mab374
1
Western Blot Analysis of EBV Proteins
2
Tau and Autophagy Protein Antibodies
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Anti–pThr-212/pSer-214/pThr-217 tau AT100 (MN1060) and anti-human tau HT7 (MN1000) were from Thermo Fisher; anti-GAPDH (MAB374) was from EMD Millipore; anti-Rab5 (ab18211), anti-p62 (ab91526), anti-NDP52 (ab68588), anti–galectin-8 (ab183637), and anti-ubiquitin (ab7780) were from Abcam; anti-p62 (ab610832) was from BD Biosciences; anti-LC3 (NB1000–2220) and anti-NDP52 (BO01P) were from Novus Biologicals; anti-optineurin (100000) was from Cayman; anti-FIP200 (17250–1-AP) was from Proteintech Group; anti-ubiquitin P4D1 (sc-8017) was from Santa Cruz Biotechnology; and anti–galectin-8 (AF1305) was from R&D Systems. Horseradish peroxidase (HRP)–conjugated secondary antibodies were from Bio-Rad. Alexa Fluor–conjugated secondary antibodies were from Life Sciences.
3
Antibody Sources and Inhibitors for O-GlcNAcylation Analysis
4
Western Blot Analysis of EMT Markers
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Western blot analysis was performed as described previously [52 (link),53 (link)]. The following antibodies were used: anti-FOXM1 (GTX100276), anti-BIRC5 (GTX100441), anti-AURKA (GTX13824), anti-CD44 (GTX102111), anti-TWIST1/2 (GTX127310), anti-ZEB1 (GTX105278), anti-CDH1 (GTX02618), anti-CK18 (GTX105624), anti-CK7 (GTX109723), and anti-VIMENTIN (GTX100619), which were obtained from GeneTex, Hsinchu City, Taiwan; anti-GAPDH (MAB374) and anti-SOX2 (AB5603), which were obtained from Merck Millipore (Burlington, MA, USA).
5
Antibody-based Western Blot Analysis
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The following antibodies used for Western blotting are listed: anti-GAPDH (MAB374, Merck Millipore, Billerica, MA, USA), anti-alpha SMA (ab5694, Abcam, Cambridge, UK), anti-vimentin (5741, Cell Signaling, Beverly, MA, USA), anti-phosphorylated AMPK (2535, Cell Signaling), anti-AMPK (2532, Cell Signaling), anti-phosphorylated mTOR (5536, Cell Signaling), anti-mTOR (ab32048, Abcam) and anti-collagen I (sc-166865, Santa Cruz, CA, USA). The chemicals used were PDGF-bb (520-BB, R&D System, Minneapolis, MN, USA), PDGF-bb(520-BB, R&D System) and n-butylidenephthalide (ALFA, Derbyshire, UK). For the in vitro experiments, 10% (w/v) n-BP/DMSO was prepared as the stock.
6
Antibodies and Inhibitors for O-GlcNAc Signaling
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Antibodies were obtained from several sources: anti-O-GlcNAc (RL-2, #MA1-072) from Pierce Biotechnology (Rockford, IL); anti-MAN1A1 (#M-3694) from Sigma-Aldrich (St. Louis, MO); anti-pAkt (ser-473, #9271s), anti-Akt, anti-pFOXO3 (ser-294, #5538s), and anti-pFOXO3 (thr-32, #9464s) from Cell Signaling (Danvers, MA); anti-Erk (K-23, #sc-94), anti-pErk (E-4, #sc-7383), anti-pIkk (T23, #sc-101706), and anti-Ikk (H470, #sc-7607) from Santa Cruz Biotechnology; anti-FOXO3 (#07–702) and anti-GAPDH (#MAB-374) from Merck Millipore (Billerica, MA). Akt inhibitor, MK-2206 dihydrochloride (#sc-364537) was from Santa Cruz Biotechnology (Santa Cruz, CA) and Erk inhibitor, PD98059 (#9900) was obtained from Cell Signaling (Danvers, MA). PNGase-F (#P0704) was purchased from New England Biolabs (Ipswich, MA), Pisum Sativum Agglutinin (PSA, #L-1050), biotinylated PSA (#B-1055), biotinylated Concanavalin A (#B-1005) were obtained from Vector Laboratories (Burlingame, CA). Mannosidase I inhibitor, kifunensine (#K1140), OGA inhibitor, [O-(2-Acetamido-2-deoxy-D-glucopyranosylidenamino)-N-phenylcarbamate; PUGNAc, #A7229], and cycloheximide (CHX, #C104450) were purchased from Sigma-Aldrich.
7
Protein Expression Analysis in Mouse Livers
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Mouse livers tissues were homogenized in lysis buffer [30 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 10% glycerol and 2 mM EDTA] containing the Complete Protease Inhibitor Cocktail (ThermoFisher Scientific, Waltham, MA, USA). Protein concentrations were determined with the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as standard. For western blotting, aliquots of 40 μg were denatured by boiling in Tris-Glycine SDS Sample Buffer (Bio-Rad), separated by SDS-PAGE, and transferred onto nitrocellulose membranes (Bio-Rad) by electroblotting. Membranes were blocked in5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h and probed with following specific antibodies for Notch2 (5732), Notch1 (3608), p-AKT (3787), total-AKT (9272) (Cell Signaling Technology Inc), and Jagged1 (Abcam, ab109536). Anti-GAPDH (MAB374, EMD Millipore, Billerica, MA, USA) and anti-β-actin (A5441, Sigma-Aldrich) were used as loading controls. Each primary antibody was followed by incubation with horseradish peroxidase-secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) diluted 1:5000 for 30 min and proteins were revealed with the Super Signal West Dura (ThermoFisher Scientific).
8
Immunoblotting Antibody Reagents and Dilutions
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Anti-Rpl1 and anti-Rpl3 were generous gifts from Jonathan Warner. Anti-FLAG (F1804; RRID:AB_262044 ; 1:10,000 dilution) was from Sigma (St. Louis, MO), anti-Hexokinase (H2035-02; 1:10,000 dilution) was from USBiological (Salem, MA), anti-HUWE1 (A300-486A; RRID: AB_2615536 ; 1:1,000 dilution) was from Bethyl laboratories (Montgomery, TX), anti-HA (SC-7392; RRID:AB_627809 ; 1:5,000 dilution) was from Santa Cruz (Dallas, TX), anti-myc (MMS-150R; RRID: AB_291325 ; 1:5,000 dilution) was from Covance (San Diego, CA), anti-Ubiquitin (05–944; RRID: AB_441944 ; 1:5,000 dilution) and anti-GAPDH (MAB374; RRID: AB_2107445 ; 1:5,000 dilution) were from EMD Millipore, and anti-His6 (200-332-382; RRID: AB_10704645 ; 1:5,000 dilution) was from Rockland (Limerick, PA). For secondary antibody, HRP-conjugated anti-rabbit IgG (A6154; RRID: AB_258284 ; 1:10,000 dilution) and HRP-conjugated anti-mouse IgG (M8770; RRID: AB_260711 ; 1:10,000 dilution) were from Sigma, IR680RD conjugated anti-rabbit (926–68071; RRID: AB_10956166 ; 1:10,000 dilution) and IR800CW conjugated anti-mouse (926–32210; RRID: AB_621842 ; 1:10,000 dilution) were from LI-COR Biosciences (Lincoln, NE).
9
Antibody Selection for PI5P4K Analysis
10
Platelet Protein Expression Analysis
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Approximately 109 platelets were lysed in 500 μl RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Deoxycolate, 0.1% SDS, 50 mM Tris HCL, pH 7.5). Proteins were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes and incubated following standard procedures. Antibodies used were Anti-Gata1 (sc-265, Santa Cruz), Anti-Syk (sc-573, Santa Cruz), Anti-Gapdh (MAB374, Merck Millipore), and secondary antibodies IRDye 680 goat anti-mouse IgG and IRDye 800CW donkey anti-mouse IgG (926–32220 and 926–32212, respectively, LI-COR Biosciences). Western blot membranes were quantified using Odyssey LI-COR Imaging system.
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