Mouse monoclonal anti gapdh

~5 OD pellets were lysed in laemmli buffer and loaded onto 10% SDS PAGE. Protein transfer was performed onto nitrocellulose membrane using cold wet tank transfer protocol. Western blotting was performed with 1:5000 dilution of mouse monoclonal anti-DBP5 and 1:10000 dilution of mouse monoclonal anti-GAPDH (Thermo Fisher) primary antibody overnight at 4°C and 1:10000 anti-mouse DyeLight 650 secondary to detect protein.

Mouse monoclonal anti-Flag M5 antibody was from Sigma-Aldrich. Rabbit anti-phospho-Smad2 and mouse anti-Myc were home-made and have been described before [59 (link)]. Mouse anti-HA was from Roche. Rabbit anti-HA and mouse anti-LKB1 used for co-immunoprecipitation and IHC assays, mouse anti-Smad1, rabbit anti-TGFβRI/ALK5 (V-22), rabbit anti-ID1, goat anti-STRADα, goat anti-Smad7, mouse anti-β-tubulin, mouse anti-ubiquitin (P4D1), and rabbit and mouse IgGs used for control immunoprecipitations, were from Santa Cruz Biotechnology. Mouse anti-BMPRIA/ALK3, mouse anti-BMPRIB/ALK6 and goat IgG used for control immunoprecipitations and immunoblotting, were from R&D Systems, Inc. Mouse monoclonal anti-GAPDH was from Ambion. Mouse anti-E-cadherin was from Becton Dickinson Transduction Labs. Rabbit anti-phospho-Smad1/5/8, rabbit anti-phospho-AMPK (Thr172), rabbit anti-AMPKα, rabbit anti-phospho-p70 S6 kinase (Thr389), rabbit anti-p70 S6 kinase and rabbit anti-ACVRI/ALK2 were from Cell Signaling Technology, while rabbit anti-MO25 and anti-Smad1 were from Epitomics, and were used for immunoprecipitation, immunoblot and IHC assays.

Proteins were extracted from PBS-washed adherent cells using NP40 buffer [20 mM Tri-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethanesulfonyl fluoride and recommended concentration of cOmplete EDTA-free protease inhibitor cocktail (Roche; one tablet per 50 ml)] and quantified using a BCA protein assay kit (Thermo Scientific). Proteins were then separated by 4–20% gradient SDS-PAGE (Bio-Rad), electrotransferred to nitrocellulose membranes and analyzed using the following primary antibodies: mouse monoclonal anti-Ptbp1 (1∶1000, Invitrogen), mouse monoclonal anti-Ptbp2 (1∶20000; a gift from R. Darnell), mouse monoclonal anti-GFP (1∶2000, Invitrogen), mouse monoclonal anti-FLAG M2 (1∶1000, Sigma), mouse monoclonal anti-Gapdh (1∶10000, Ambion). Immunoblot signals were visualized using corresponding secondary antibodies conjugated with horseradish peroxidase (GE Healthcare) and Immobilon Western ECL reagents (Milipore).

Lysates (30-50 μg) were prepared from the carotid artery, aorta or VSMCs, subjected to SDS-polyacrylamide gel (10%) electrophoresis and transferred to a PVDF membrane (Perkin Elmer Life Sciences, Boston, MA) [16 ]. Antibodies used include a mouse monoclonal anti-nestin (1:500; Chemicon), mouse monoclonal anti-eNOS (1:500; BD Bioscience), goat monoclonal anti-CD31 (1:500; SantaCruz Biotechnologies), rabbit polyclonal anti-caldesmon (1:2500; Abcam), rabbit polyclonal anti-smooth muscle-22α (1:5000; Abcam), rabbit polyclonal anti-smooth muscle α-actin (1:5000; Abcam), and mouse monoclonal anti-GAPDH (1:50,000; Ambion, Austin TX). Following overnight incubation at 4°C, the appropriate secondary antibody-conjugated to horseradish peroxidase (1:20,000, Jackson Immunoresearch, West Grove, PA) was added and bands visualized utilizing the ECL detection kit (Perkin Elmer). Films were scanned with Image J software® and the target protein signal was depicted as arbitrary light units normalized to GAPDH protein levels.

Lysates (30–50 μg) prepared from the left ventricle of normal and suprarenal aorta constricted rats, adult and neonatal ventricular fibroblasts and rat microvascular endothelial cells were subjected to SDS-polyacrylamide gel (15%) electrophoresis and transferred to a PVDF membrane (Perkin Elmer Life Sciences, Boston, MA).[6 (link),14 (link)] Antibodies used include mouse monoclonal anti-nestin (~240 KDa;1:500; EMD Millipore, Darmstadt, Germany), goat monoclonal anti-CD31 (~140 kDa; 1:500; Santa Cruz Biotechnologies, Santa Cruz, CA), mouse monoclonal anti-smooth muscle α-actin (~43 kDa; 1:5000; Sigma-Aldrich), rabbit polyclonal anti-collagen type I (~240 KDa pro-collagen;1:1000; Abcam, Cambridge, MA), rabbit polyclonal anti-prolyl 4-hydroxylase alpha polypeptide III (~75 KDa;1:500; EnoGene, New York, NY) and a mouse monoclonal anti-GAPDH (~37 kDa; 1:50,000; Ambion, Austin TX). Following overnight incubation at 4°C, the appropriate secondary antibody-conjugated to horseradish peroxidase (1:20,000, Jackson Immunoresearch, West Grove, PA) was added and bands visualized utilizing the ECL detection kit (Perkin Elmer). Films were scanned with Image J software^® and the target protein signal was depicted as arbitrary light units normalized to GAPDH protein.

Total protein was isolated from transfected cells with a SDS-based lysis buffer followed by protein concentration determination using the Bio-Rad DC^TM Protein assay as per the manufacturers’ protocol (Bio-Rad Laboratories). Thirty micrograms of protein was loaded onto a 12% Sodium Dodecyl Sulfate Polyacrylamide (SDS-PAGE) gel and the subsequent resolved gel was transferred to a nitrocellulose membrane (Amersham Protran 0.45 NC). Membranes were blocked with 5% skim milk before exposure to primary antibody: 1:1000 anti-GAPDH mouse monoclonal (Invitrogen 39-8600); 1:100 anti-HBsAg mouse monoclonal (Santa Cruz sc-53299); 1:500 anti-HBcAg mouse monoclonal (Abcam ab8637); 1:1000 anti-APOBEC3G rabbit polyclonal (Abclonal #A1459). Secondary antibody used was anti-mouse IgG HRP conjugated (GE healthcare NA-931) at 1:10000 (for GAPDH) and 1:1000 (for anti-HBsAg and anti-HBcAg) or anti-rabbit IgG HRP conjugated (GE healthcare NA-934) at 1:1000 (for APOBEC3G). Blots were developed with Immobilon^® Forte Western HRP substrate (Millipore Sigma). Densitometry analysis was performed using the Image Studio^TM Lite software version 5.2 (LI-COR Biosciences, Lincoln, United Kingdom).