Image lab 6.0 software chemidoc

To assess the level of activated p38, JNK, and Akt, wt or transfected cells were starved 18 hours before lysis. Whole cell lysates were subjected to SDS-PAGE, transferred to PVDF membrane and the levels of activated p38, JNK and Akt were detected in Western blot by using phospho-specific monoclonal antibodies against P-p38 and P-JNK and polyclonal antibodies against P-Akt (Cell Signaling Technology). To detect the total amount of proteins loaded, the blot was re-probed with polyclonal antibodies against p38, JNK (Santa Cruz Biotechnology), and Akt (Cell Signaling Technology). Bands signal intensity was measured by densitometry using Image Lab 6.0 software (ChemiDoc, Bio-Rad) and normalized to each total protein expression.

Protein lysates were obtained by lysing cells in Staph-A buffer (1.6 mM NaH2PO4; 8.6 mM Na2HPO4; 1% Triton X-100; 0.1% SDS; 0.1% NaCl; 0.5% NaDoc; 2 mM AEBSF; 20 mg/mL each of aprotinin and leupeptin). Proteins (100 µg) were subjected to SDS-PAGE electrophoresis, transferred to PVDF membrane (Millipore), and then blots were probed with the primary antibodies, followed by the HRP-conjugated secondary antibodies raised in mouse or rabbit (ThermoFisher Scientific). Anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology) was used as loading control. Bands visualization was performed by ECL Select Western Blotting Detection Reagent (Amersham, GE Healthcare, Little Chalfont, UK), then signal intensity was measured by densitometry using Image Lab 6.0 software (ChemiDoc, Bio-Rad, Hercules, CA, USA) and normalized to the loading control⁵⁰ .