Goat anti rabbit igg h l

All chemicals and solvents used in the present study were of highest grade available. InuA with a purity higher than 95% was prepared as reported previously [34 (link)]. The antibodies against human MDM2 (Ab-2), p21 (Ab-1), and p-H2AX (Ser139) were bought from EMD Chemicals (Gibbstown, NJ, USA). The antibodies against human p53 (DO-1), Cdk2 (M2), Cdk4 (H-22), Cdk6 (C-21), cyclin D1 (DCS-6), cyclin E (HE12), c-Myc (0.N.222), Bax (N-20), Bcl-2 (100), PARP (H-250), Chk1 (G-4), Chk2 (B-4), and ATR (N-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against human p-Chk1 (Ser317), p-Chk2 (Thr68), and p-p53 (Ser15) were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-human NFAT1 (1/NFAT-1) antibody was sourced from BD Biosciences (San Jose, CA, USA) and the anti-human β-actin (AC-15) antibody was purchased from Sigma (St. Louis, MO, USA). The goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) antibodies were bought from Bio-Rad (Hercules, CA, USA). The NFAT1 and MDM2 siRNA pool and control siRNA pool were obtained from Thermo Scientific (Rockford, IL, USA).

Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris- HCl, pH 8.0) supplemented with protease Inhibitor Complete Mini (Roche) on ice for 15 min. The cell lysates were rotated at 4°C for 30 min and the insoluble material was removed by centrifugation at 14,000 rpm for 15 min. Protein concentrations were determined by BioRad Protein Assay (BioRad) using bovine serum albumin (BSA) as a standard. Following the normalization of protein concentrations, lysates were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95°C prior to separation by SDS PAGE on stain free TGX gradient gels (BioRad). Following SDS PAGE, the proteins were transferred to polyvinylidene fluoride membranes (300 mA for 90 min at 4°C). The membranes were then blocked with BCA (SIGMA) proteins dissolved in PBS/Tween-20 (3% BCA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody specified for each experiment (Bax abcam ab32503; BCL2 abcam ab59348, beta actin abcam ab1801, and caspase 8 santa cruz sc-81661). After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H+L) or with goat anti-mouse IgG (H+L) HRP-conjugated secondary antibodies (BioRad) and detected using ECL (Pierce). Densitometry was performed using Image Lab software v. 4.1 (BioRad).