Igg hrp linked secondary antibodies

Whole cell protein extracts were prepared from 4.5×10⁶cells using 50μL lysis buffer containing 20mM Tris(hydroxymethyl)aminomethane (Tris) pH 7.4, 1mM EDTA, 140mM NaCl, 1% NP-40 supplemented with 2mM sodium vanadate and protease inhibitor cocktail (Sigma-Aldrich, San Louis, MO, USA) for 1 hour at 4°C. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of denatured protein were resolved by 10% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Membranes were blocked for 1 hour at room temperature (RT) in 5% milk/TBS-T. Membranes were incubated overnight at 4°C with primary antibodies against ZAP-70, Mcl-1 and Bcl-2 (Santa Cruz Biotechnologies, Dallas, TX, USA), and GAPDH (Abcam, Cambridge, United Kingdom). Immunodetection was done with the corresponding IgG HRP-linked secondary antibodies (Dako North America, Glostrup, Denmark), and the ECL chemiluminescence detection system (GE Healthcare). Chemiluminescent images were acquired with the LAS-4000 system (Buckinghamshire, United Kingdom). Protein expression was subsequently quantified using the Image J 1.46r software (National Institutes of Health).

BCR was stimulated with 5 µg/mL F(ab)₂ anti-IgM (Invitrogen). ERK1/2 and Akt were inhibited with PD98059 (Cell Signaling Technology, Inc.) and LY294002 (Sigma-Aldrich), respectively. Protein from cell lines was extracted using 20 mM Tris pH 7.4, 1 mM EDTA, 140 mM NaCl, 1% of NP-40 supplemented with 2 mM sodium vanadate and protease inhibitor cocktail (Sigma-Aldrich) for 30 minutes at 4 °C. Protein concentration was determined using the Bio Rad Protein Assay system (Bio-Rad). Equal amounts of denatured protein were separated by 10% poliacrilamide-SDS gel electrophoresis. The size-separated proteins were transferred to Immobilon-phosphate membranes (Millipore). Membranes were blocked with 5% milk/TBS-T (Tris-Buffered Saline Tween-20) for 1 hour at room temperature (RT). Membranes were incubated overnight at 4 °C with primary antibodies against phospho-ZAP-70^Tyr319/Syk^Tyr352, phospho-Akt^Ser473, phospho-ERK1/2^{Thr202/Tyr204}, phospho-STAT3^Ser727, Akt, ERK1/2, PTEN and PDCD4 (Cell signalling Technology Inc.), ZAP-70 (clone 2F3.2; Upstate Biotechnology), PIAS3 (WuXi AppTec.ABGENT) and β-actin (Abcam). Immunodetection was done with the corresponding IgG HRP-linked secondary antibodies (Dako North America) for 1 hour at RT. Chemiluminescent images were obtained using LAS-4000 equipment (Buckinghamshire).