Mouse monoclonal anti gapdh mab374

The cells were lysed 48 h posttransfection in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.2% sodium dodecyl sulfate, 50 mM Tris-HCl, 1× Protease inhibitor cocktail Complete (Roche), and 1 mM DTT). The protein concentrations were measured with Pierce BCA Protein Assay Kit (Thermo Scientific).
Equal amounts of protein were separated in 8 to 10% SDS-polyacrylamide gel and transferred to Immobilon-P polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% skimmed milk in PBS-0.1% Tween 20 (PBST) (Sigma-Aldrich) at room temperature and incubated with primary and secondary antibody solutions in 2% skimmed milk in PBST. The antibodies were used in the following dilutions: rabbit polyclonal anti-TCF4 (CeMines) 1:1000, mouse monoclonal anti-E2 (5E11, Icosagen,) 1:5000, mouse monoclonal anti-GAPDH (MAB374, Millipore) 1:4000, horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG (Thermo Scientific) 1:5000. For chemiluminescent reaction, SuperSignal West Femto Maximum Sensitivity Substrate Kit (Thermo Scientific) was used, and the reaction was visualized with ImageQuant LAS4000 camera system (GE Healthcare).

For western blot analysis, the following primary antibodies were used: mouse monoclonal anti-GFAP (ab4648; Abcam, Cambridge, UK), mouse monoclonal anti-Tuj1 (MMS435; Covance, Princeton, NJ, USA), mouse monoclonal anti-P53 (2524; Cell Signaling Technology Inc., Boston, MA, USA), rabbit monoclonal anti-p-CREB (9198; Cell Signaling Technology Inc.), rabbit monoclonal anti-p-PKA (5661; Cell Signaling Technology Inc.) and mouse monoclonal anti-GAPDH (MAB374; Millipore, Billerica, MA, USA).