Beta actin mouse monoclonal antibody

Protein extracts were prepared from tissue samples (∼50 mg) by homogenization in 500 μl whole cell lysis buffer (1% NP-40, 10 mM Tris pH 7.8, 150 mM NaCl, 40 mM EDTA, 10 mM Na-Pyrophosphate, 10 mM NaF, 1mM PMSF, 4 mM Orthovanadate, Minicomplete iprotease inhibitor^®). Protein extracts (30 μg) were separated by polyacrylamide gel electrophoresis. Blottings were incubated one hour at RT in a blocking buffer containing 5% skim milk and then incubated overnight at 4°C with mouse monoclonal anti-phosphorylated STAT3 (Santa Cruz), rabbit monoclonal anti phosphorylated AKT (Ser473) (Cell Signaling, Danvers, MA), mouse monoclonal anti Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), mouse monoclonal anti Diphosphorylated-JNK (JNK-PT48) (Sigma) and beta-actin mouse monoclonal antibody (Sigma-Aldrich), and subsequently, with peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (Dako) for one hour at room temperature.

Unless specified, the primary antibody to Spns2 was a goat polyclonal antibody directed against an intracellular domain (G-14, Santa Cruz, Dallas, TX, USA). In additional experiments, rabbit polyclonal antibodies directed against the aa71-120 domain (LifeSpan BioSciences, Seattle, WA, USA) or the N-terminus (GeneTex, Irvin, CA, USA) were used to confirm similar patterns of immunofluorescence. Rabbit SPHK1 and SPHK2 polyclonal antibodies were from Bioss (Woburn, MA, USA). Beta-actin mouse monoclonal antibody was from Sigma-Aldrich (St. Louis, MO, USA). PAN (rabbit anti-cytokeratin) antibody was from Invitrogen (Carlsbad, CA, USA). Secondary donkey IgG F(ab’)2 fragment antibodies conjugated to AF488, AF594 or AF647, specific to rabbit, goat, and mouse IgG (respectively) were all from Jackson ImmunoResearch (West Grove, PA, USA).

Freshly isolated guinea pig ventricular myocytes were cultured for 24 h at 37°C in M199 medium under control conditions or with KChIP2 antisense virus. Upon collection, cardiomyocytes were washed in ice-cold PBS and resuspended in a RIPA lysis buffer (150 mM sodium chloride, 1.0% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl sulphate), 50 mM Tris, pH 8.0, plus Roche Inhibitor tablet) and then sonicated on ice to disrupt cell membranes. 30–40 μg of whole cell extract was loaded into SDS-PAGE gels, transferred to nitrocellulose membrane, and Western blotting performed using KChIP2 mouse monoclonal antibody (UC Davis NeuroMab 75–017) at a 1:500 dilution, beta-actin mouse monoclonal antibody (Sigma-Aldrich, A1978) at a 1:1000 dilution, Cav1.2 mouse monoclonal antibody (UC Davis NeuroMab 75–257) at a 1:500 dilution, Nav1.5 mouse monoclonal antibody (Sigma S8809) at a 1:800 dilution, and pan-cadherin rabbit monoclonal antibody (Cell Signaling 4068S) at a 1:1000 dilution. Protein concentrations were determined by the BCA method (Pierce).