Molecular imager gel doc xrs system

Primary antibodies against HSP90 (C45G5) and β-actin were purchased from Cell Signaling Technology. LC3B antibody was from GenTex. HSC70 (13D3) was purchased from Abcam. The anti-p53 antibody (DO-1) was a gift from Dr Xirodimas. Cell extracts were lysed in 2x SDS sample buffer. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose or PVDF membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Peroxidase-coupled anti-mouse and anti-rabbit secondary antibodies were used at a dilution of 1:10.000 (Sigma). Bound antibodies were detected by enhanced chemiluminescence (Millipore). To obtain the image for the western blotting the Molecular Imager Gel Doc XRS system (Biorad) was used, this system provides a reliable and sensitive imaging of chemilunescence western blots. For the quantification and analysis, we used the Image Lab Software (Biorad), a powerful and specific software for acquisition, analysis and quantification of blot images.

Primary antibodies against phospho-AMPKα (Thr172), AMPKα, AMPKβ, phospho-ACC (Ser79), ACC, phospho-p53 (S15 and S46), p21 (12D1) and β-actin were from Cell Signaling Technology. The anti-p53 antibody (DO-1) was a gift from Dr Xirodimas. Cell extracts were lysed in 2 × SDS sample buffer. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose or PVDF membranes using the Trans-Blot^® Turbo™ Transfer System (Bio-Rad). Peroxidase-coupled anti-mouse and anti-rabbit secondary antibodies were used at a dilution of 1:10, 000 (Sigma). Bound antibodies were detected by enhanced chemiluminescence (Millipore) or using the Supersignal West Dura extended duration substrate (Pierce). Chemiluminescent detection was carried out using the Molecular Imager Gel Doc XRS system (Bio-Rad). Band quantification was performed using the Image lab software (Bio-Rad). The P-ACC/ACC ratio is calculated by given the value of 1 to untreated cells for each independent cell line and reporting to this value the P-ACC/ACC ratio in stimulated cells.