β actin hrp c 4

In order to prepare whole cell lysates, cells were washed with ice-cold phosphate-buffered saline (PBS; Biowest, Nuaillé, France) and lysed with ice-cold Denaturing Cell Extraction Buffer (FNN00091, Thermo Fisher Scientific, Waltham, MA, USA), incubated on ice for 30 min, and centrifuged for 15 min at 4 °C. The supernatant was used for protein content determination and subsequent immunoblotting. For immunoblotting standard procedures using the following antibodies were used as previously described (Haas et al. 2009 (link)). Anti-MOR-1 (D-12; 1:1000, Santa Cruz Biotechnology, TX, USA) combined with goat antimouse IgG-HRP (1:5000, Santa Cruz Biotechnology, TX, USA) and β-Actin-HRP (C-4; Santa Cruz Biotechnology, TX, USA). Immunoblots were developed with the enhanced chemoluminescence system (Amersham Biosciences, Little Chalfont, United Kingdom).

For immunoblotting standard procedures using the following antibodies were used as previously described [53 (link), 54 (link)]. Anti-EGF-R (AF-231), anti-IGF-R (AF-305-NA), anti-PDGF-Rβ (AF-385) combined with donkey anti-goat IgG-HRP (HAF 109; all R&D systems). Anti-MRP-2 (M2 III-6), Anti-P-gp (D-11; both Santa Cruz Biotechnology) combined with goat anti-mouse IgG (H + L)-HRP (Thermo Scientific). Anti-P53-phospho-S15 (A7180; Assay Biotechnology Company), P21 (C-19, Santa Cruz Biotechnology) combined with goat anti-rabbit IgG (H + L)-HRP (Thermo Scientific). P53-HRP (DO-1) and β-Actin-HRP (C-4; both Santa Cruz Biotechnology). Immunoblots were developed with the enhanced chemoluminescence system (Amersham Biosciences).

Cells were lysed in 1% NP40 buffer (1% NP40, 10% glycerol, 20 mM Tris–HCl (pH7.5), 150 mM NaCl, 1 mM EGTA) complemented with complete protease inhibitors (#11697498001, Roche Biochemicals, Basel, Switzerland) and 1 mM PMSF (Roth, Karlsruhe, Germany) and incubated on ice for 30 min. The lysates were clarified by centrifugation for 15 min at top speed and eluted in SDS-PAGE sample buffer. SDS-PAGE was performed using Novex 4–12% gels (Life Technologies, Carlsbad, CA) with MES running buffer (Thermo Fisher Scientific, Waltham, MA) and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Kenilworth, NJ). The membranes were blocked in 5% skim milk plus Tris-buffered-saline-Tween 20 (TBST) at room temperature for 1 h and then incubated in primary antibody overnight at 4 °C. Primary antibodies used are α-NLRC4 (rabbit anti-human NLRC4, D5Y8E, Cell Signalling Technologies, Danvers, MA), β-Actin-HRP (C4) (sc-47778, Santa Cruz Biotechnology, Dallas, TX), and monoclonal anti-FLAG M2-peroxidase (HRP) antibody (Sigma-Aldrich, St. Louis, MO).