Anti β actin c 11

Total protein extracts were prepared as previously reported [27 ]. Immunoprecipitation and immunoblotting were performed accordingly to standard procedures [27 ]. The Abs employed for the study were as follows: anti-IL-22R1, anti-phospho-STAT3 (Ser727), anti-STAT3 (C-20), anti-phospho-STAT1 (Tyr701), anti-STAT1 (E-23), anti-phospho-Erk1/2 (E-4), anti-Erk1/2 (C16), anti-β-actin (C-11), and HRP-conjugated anti-c-myc (9E10), all provided by Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-STAT3 (Tyr705) anti-MEK (mitogen-activated protein kinase) 1/2 and anti-phospho MEK1/2 (Ser218/222) were from Cell Signaling Technology (Denvers, MA); anti-SOCS1 and anti-SOCS3 were from MBL (Sigma-Aldrich, Milan, Italy), whereas anti-JAK1 and anti-phospho Tyr were from Upstate (Millipore, Milan, Italy). Filters were properly developed with anti-mouse, anti-goat, or anti-rabbit Ig Abs conjugated to HRP using the ECL-plus detection system (Amersham, Dubendorf, Switzerland), or, otherwise, the SuperSignal West Femto kit (Pierce, Rockford, IL, USA).

Protein lysates were separated by SDS-PAGE, transferred, and probed with primary and secondary antibodies as described [19 (link)]. For the analysis of cytochrome-c and AIF, cytosolic and mitochondrial sub-cellular fractions were separated using the mitochondria isolation Kit for Cultured Cells (Thermo Scientific, Rockford, IL, USA). To ensure equivalent protein loading and transfer, all blots were probed for β-actin. Unless otherwise indicated, all Western blots shown in the figures are representative of three independent experiments. Membrane probing included the following primary antibodies: anti–human TCL1 (clone 1–21) [19 (link)]; anti-BCL2 (C-2), anti-AIF (D-20), and anti-β-actin (C-11) from Santa Cruz Biotechnology, Texas, USA; anti-phospho (p)-P53 (Ser15), anti-P53 (1C12), anti-caspase-3, anti-cytochrome c, and anti-PARP from Cell Signaling Technology, Massachusetts, USA.; anti-Smac/Diablo from BD Transduction Laboratories, Germany; and anti-human LC3B from Novus Biologicals, Colorado, USA. The HRP-conjugated species-specific secondary antibodies were from Dianova, Germany. Band detection by chemiluminescence (Amersham Buchler, Germany) was followed by densitometry by ImageJ (rsb.info.nih.gov/ij), including normalization to β-actin expression.