Mcl 1 clone s 19

Whole cell protein extracts were obtained by lysing 2–5 × 10⁶ cells with Laemmli sample buffer. Protein concentration was measured with the Micro BCA^TM Protein Assay Reagent kit (Thermo Scientific Pierce, Rockford, IL, USA). Protein extracts (25 μg) were subjected to reducing conditions before being subjected to electrophoresis on a polyacrylamide gel and then transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). One hour after blocking with 5% (w/v) non-fat milk in Tris-buffered saline with 0.1% Tween^®-20, the membranes were incubated with the specific primary antibodies against BCL-2 (clone 124, #M0887, Dako, Denmark), BIM (clone C34C5, #2933, Cell Signaling, Danvers, MA, USA), cleaved caspase 3 (#9661, Cell Signaling), ERK2 (clone 1B3B9, #05-157, Millipore, Temecula, CA, USA), NOXA (clone 114C307, #ab13654, Abcam, Cambridge, UK), MCL-1 (clone S-19, #sc-819, Santa Cruz Biotechnology, Dallas, TX, USA), PHB1 (clone H-80, #sc-28259, Santa Cruz Biotechnology), PHB2 (anti-REA, #07-234, Millipore), PARP (#9542, Cell Signaling), PUMA (#4976, Cell Signaling), β-Actin (clone AC-15, #A5441, Sigma-Aldrich) and Tubulin (clone Ab-1, #CP06, Oncogene, Darmstadt, Germany). Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Amersham Place, Buckinghamshire, UK).

After treatment, cells were washed twice with ice-cold PBS containing 10% fetal bovine serum, centrifuged at 1000 r.p.m. for 5 min, and lysed in 50 μl of ice-cold Cell Lytic (Sigma) supplemented with protease (Roche Diagnostics Corporation, Indianapolis, IN, USA) and phosphatase (Sigma) inhibitors. Protein concentrations were determined by the BSA assay (Invitrogen) and 50 μg of protein electrophoresed by SDS–PAGE (Invitrogen). Separated proteins were transferred to nitrocellulose membranes utilizing an iBlot (Invitrogen) device. Blots were probed with MCL-1 (clone S-19; Santa Cruz Biotechnology, La Jolla, CA, USA), PARP (clone C2-10) and BCL-2 (Clone 7; both BD Biosciences, CA, USA), caspase-3 (clone 31A1067, Abcam, Cambridge, UK) or β-actin (Sigma) antibodies followed by IRDye 680/800CW-conjugated secondary antibodies (LICOR Biosciences, NE, USA). Proteins were visualized using the Odyssey infrared imaging system (LICOR Biosciences) and were not further manipulated with imaging software.

Cells were lysed in M-PER buffer (Pierce Biotechnology, Rockford, IL, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma). Total cell extracts were separated using SDS-PAGE in 10% gels, transferred to nitrocellulose membranes, and blotted as described previously [97 (link)]. The primary antibodies included anti-NGAL (clone AF1757, goat IgG, specific for the dimeric and monomeric forms; R&D Systems), anti-MMP-9 (clone EP1254, rabbit Ig, specific for CPX; Abcam, Paris, France), anti-NGAL-R/Slc22A17 (clone 75010, rabbit Ig; Biorbyt), and antibodies against Mcl-1 (clone S-19, rabbit IgG; Santa-Cruz), Bcl-2 (clone 100, mouse IgG1; Santa-Cruz), STAT3 (clone C20, rabbit IgG; Santa-Cruz), phospho-Tyr⁷⁰⁵-STAT3 (clone 710093, rabbit Ig; Thermo Fisher Scientific, Waltham, MA, USA), STAT1 (clone -23, rabbit IgG; Santa-Cruz) and actin (clone C4, mouse IgG1; ICN Biomedicals, Solon, OH, USA). Immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary antibodies and visualized with the Pierce ECL Western blotting substrate system or the SuperSignal West Femto Maximum Sensitivity Substrate system (both from Thermo Fisher Scientific). Immunoblot images were acquired in an MF-ChemiBIS 4.2 imager (DNR Bio-Imaging Systems Ltd., Neve Yamin, Israel) and quantified using ImageJ64 software.