Phospho p38

Cells were lysed on ice for 30 min with RIPA lysis buffer, which contains 50 mM Tris^_HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml aprotinin). For western blotting, 25 μg of protein sample was resolved on 12.5% SDS-PAGE and electrotransferred onto nitrocellulose membranes (Whatman). The primary antibodies used were as follows: phospho-Akt, total Akt, phospo-p44/p42 ERK, total p44/p42 ERK, phospho-p38, total-p38, phospo-JNK, and total-JNK were purchased from Cell Signaling Technology (1:1000 dilution ratio). Anti-NFATC1 antibody was purchased from Santa Cruz (1:500 dilution ratio). β-actin or GAPDH was used as loading control. Horseradish peroxidase-conjugated secondary antibodies were used at a 1:5000 dilution. The antigen–antibody complexes were visualized using the enhanced chemiluminescence detection system (Millipore) following the manufacturer’s instructions. Immunoreactive bands were quantitatively analyzed in triplicate by normalizing the band intensities to their respective controls on scanned films using ImageJ software.

A cell counting kit (CCK) for WST assay was purchased from Donginbiotech Co. (Seoul, Korea). Crystal violet solution was purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-phospho-AMPK, phospho-p38, phospho-ERK, phospho-JNK, cyclin D1, CDK4, AMPK, PARP, caspase-3, Bcl-2, Bcl-xL, and Bax antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p38, ERK, JNK, GAPDH, and α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).