HepG2 cells were grown in 100 mm culture dishes with or without LC combined or not with F. Cellular extracts were obtained lysing the cells in RIPA buffer as previously described [24 (link)]. Thirty micrograms of proteins were separated by SDS–polyacrylamide gel electrophoreses (SDS-PAGE) and electrophoretically transferred to nitrocellulose membranes (Potran®, Whatman® Schleicher & Schuell). The blots were then blocked and probed with specific primary antibodies, followed by incubation with anti-species-specific secondary antibodies.
To confirm equal protein loading per sample, we used antibody anti-calnexin or anti-GAPDH. Detection of specific proteins was performed by enhanced chemioluminescence reagent (Western Lightning ECL Pro, Perkin Elmer). Quantitative measurement of immunoreactive band intensities was performed by densitometry analysis using the Scion Image software (Scion Corporation, Frederick, MD, USA). Only for AMPK inhibitor experiments and CaMKII, immunoreactive bands were visualized by Uvitec Alliance LD9 gel imaging system (Uvitec, Cambridge, UK).
Data were then presented as ratio between treated cells and control.
To confirm equal protein loading per sample, we used antibody anti-calnexin or anti-GAPDH. Detection of specific proteins was performed by enhanced chemioluminescence reagent (Western Lightning ECL Pro, Perkin Elmer). Quantitative measurement of immunoreactive band intensities was performed by densitometry analysis using the Scion Image software (Scion Corporation, Frederick, MD, USA). Only for AMPK inhibitor experiments and CaMKII, immunoreactive bands were visualized by Uvitec Alliance LD9 gel imaging system (Uvitec, Cambridge, UK).
Data were then presented as ratio between treated cells and control.
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