Rabbit anti mouse polyclonal igg to β tubulin

Prostates were lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 5 mM NaF, 2 mM sodium orthovanadate, 1 mM PMSF, 1× complete protease inhibitors), boiled at 100°C for 10 min in Laemmli buffer (BioRad) with 2-mercaptoethanol, and run on a 4–20% gradient SDS-PAGE gel (BioRad). Following transfer to a nitrocellulose membrane, blots were probed with rabbit anti-mouse mAbs to CREB or pCREB (Cat #9197S and 9198, respectively, Cell Signaling), rabbit anti-mouse polyclonal Ab to pPKA C (Cat #4781S, Cell Signaling) or rabbit anti-mouse polyclonal IgG to β-tubulin (Cat #2146S, Cell Signaling) followed with mouse anti-rabbit HRP conjugate (Cat #SC-2357, Santa Cruz), and developed using an ECL plus chemiluminescence kit (Cat#32132, Thermo Fisher Scientific). B16-F10 melanoma cells (Cat#CRL-6475, ATCC) treated 1 h with or without 25 μM forskolin (FSK, adenylyl cyclase activator) served as pCREB positive and negative controls, respectively. Protein quantification was performed using GeneTools software (Syngene).