Anti actin sc 1616 r

Cells were lysed in buffer containing 1% Nonidet P-40, 1 mmol/liter EDTA, 50 mmol/liter Tris-HCl (pH 7.5), and 150 mmol/liter NaCl supplemented with Complete protease inhibitors (Roche). Total proteins were resolved in a 10-12% polyacrylamide gel under denaturing conditions and transferred to nitrocellulose filters for Western blot analyses. Membranes were blocked with 5% BSA in TBS and incubated with the primary antibodies. Membranes were then incubated with the horseradish peroxidase–conjugated secondary antibody (1:3.000) and the reaction was detected with a Western blotting detection system (enhanced chemiluminescence; GE Healthcare). The primary antibodies used were: anti-PATZ1 [22 (link)]; anti-NESTIN (sc-23927, Santa Cruz Biotechnology, Santa Cruz, CA); anti-PDGFRβ (3169, Cell Signaling Technology, Boston, MA); anti-NANOG (sc-134218, Santa Cruz Biotechnology); anti-SOX2 (sc-20088, Santa Cruz Biotechnology); anti-CXCR4 (C8727, Sigma-Aldrich). To ascertain that equal amounts of protein were loaded, the membranes were incubated with anti-ACTIN (sc-1616-R, Santa Cruz Biotechnology) or anti-VINCULIN (sc-7649, Santa Cruz Biotechnology) antibodies.

Cell pellet preparation and western Blot analysis were performed as previously described [27 (link)]. The membrane was incubated with one of the following primary antibodies: anti-E2F1 (sc-251, Santa Cruz Biotechnology); anti-Actin (sc-1616r; Santa Cruz Biotechnology); anti-SPNS2 (catalog no. SAB1304232).