Horseradish peroxidase hrp conjugated goat anti mouse

Immunoblotting was carried out as previously described (20 (link)). For VEGF stimulation, 6-well plates with cells at 70% confluency were incubated in low serum conditions (0.2% FBS) ± cediranib (100 nM) overnight and then treated with VEGF (0–100 ng/ml) for 0–60 min. Protein was extracted using 50 mM Tris-HCl pH 7.6, 137 mM NaCl, 10% glycerol, 0.1% Igepal, 0.1% SDS, 50 mM NaF, 1 mM Na₃VO₄ and cocktail protease inhibitor (1 tab per 25 ml of lysis buffer) on ice for 10 min. Immunoblots were incubated overnight at 4°C with antibodies against total and phosphorylated VEGFR2 (CST, Hertfordshire, UK. 1:800 dilution), p42/44 MAPK (CST, 1:1,000 dilution), AKT (CST, 1:1,000 dilution), PARP (CST, 1:1,000 dilution) and PPIB (Abcam, Cambridge, UK; 1:2,000). After washing, blots were incubated for 40 min with anti-mouse or anti-rabbit LI-COR secondary antibodies (LI-COR, Cambridge, UK; 1:50,000 dilution) or with horse-radish peroxidase (HRP) conjugated goat anti-mouse (Thermo Scientific, Boston, MA, USA; 1:8,000 dilution) or anti-rabbit (Thermo Scientific; 1:6,000 dilution) for 1 h. Detection was carried out using the LI-COR, Odyssey or ECL substrate (Thermo Scientific).

GA101 was kindly provided by Roche Glycart AG. Thapsigargin and tunicamycin were purchased from Merck Millipore (Fontenay s/s bois, France). BTP2, Ned-19 trans, and puromycin were supplied by Tocris Bioscience (Lille, France). Hoechst 33258 and siramesine were purchased from Sigma-Aldrich (L’Isle d’Abeau, France). Tetramethylrhodamine methyl ester (TMRM) and lysotracker red DND-99 were from ThermoFisher Scientific (Courtaboeuf, France), and Fluo2-leak resistant (LR)- acetoxymethyl ester (AM) was from Euromedex (Mundolsheim, France). FAM-FLICA in vitro caspase 3 detection kits were supplied by AbD Serotec (Kidlington, UK).
Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel). The anti-human CD19-PE, CD19-488 (clone HIB19), and anti-human CD20-FITC (clone 2H7) and their respective isotype controls were provided by eBiosciences (San Diego, CA, USA). Anti-cathepsin B and anti-BIM were supplied by Santa Cruz Biotechnology (Heidelberg, Germany). The anti-phospho eIF2α and anti-eIF2α were from Cell Signaling Technology (Ozyme, France). Alexa 594-conjugated donkey anti-goat came from Life Technologies (Saint Aubin, France). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit were from ThermoFisher Scientific.