Cells were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors and centrifuged at 15,000 g for 15 min at 4 °C. Protein concentration was determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA), and equal amounts of protein (20–30 μg) were subjected to 8–12% SDS-PAGE and transferred to an Immobilon membrane (Millipore, Bedford, CA, USA). Blots were treated with primary antibodies, followed by the corresponding secondary antibody with horseradish peroxidase activity. Blots were developed using Pierce ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA) in a Fujifilm LAS 3000 Intelligent Dark Box IV imaging system (Tokyo, Japan).
The following primary antibodies were used: anti-PEPCK-M (ab70359, Abcam, Cambridge, UK), anti-PEPCK-C (generous gift of Dr. Daryl K. Granner, Vanderbilt University, Nashville, TN, USA), anti-SOD2 (ab13534, Abcam), anti-p53 (ab26, Abcam), anti-p21 (sc397, Santa Cruz Biotechnology, Dallas, TX, USA), anti-gamma tubulin (T6557, Sigma-Aldrich, St. Louis, MO, USA), and anti-PKC-ζ (9372S, Cell Signaling Technology, Danver, MA, USA).
The following primary antibodies were used: anti-PEPCK-M (ab70359, Abcam, Cambridge, UK), anti-PEPCK-C (generous gift of Dr. Daryl K. Granner, Vanderbilt University, Nashville, TN, USA), anti-SOD2 (ab13534, Abcam), anti-p53 (ab26, Abcam), anti-p21 (sc397, Santa Cruz Biotechnology, Dallas, TX, USA), anti-gamma tubulin (T6557, Sigma-Aldrich, St. Louis, MO, USA), and anti-PKC-ζ (9372S, Cell Signaling Technology, Danver, MA, USA).