Hrp conjugated anti rabbit or anti mouse igg antibody

The protein levels of AKT, phosphorylated AKT, BCL2, BAK and BAX were determined by Western immunoblotting analysis as previously described [34 (link)]. In brief, proteins isolated from cells (50 μg) were loaded and separated by electrophoresis on a 10% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was probed with either rabbit anti-Akt antibody (Cell Signaling Technology), rabbit anti-phosphorylated AKT antibody (Cell Signaling Technology), rabbit anti-BAK antibody (Cell Signaling Technology), rabbit anti-BAX antibody (Cell Signaling Technology), or mouse anti-BCL2 antibody (Neomarkers). All antibodies were diluted at 1:1000. HRP-conjugated anti-rabbit or anti-mouse IgG antibody (Jackson ImmunoResearch) was used as the secondary antibody. The corresponding protein bands were visualized using enhanced chemiluminescence reagents. The same membranes were re-probed with rabbit anti-β-actin monoclonal antibody (Abcam) to confirm equal loading of proteins for each sample.

Yap protein levels were determined by western immunoblotting analysis. In brief, proteins isolated from cells (40 μg) were separated by electrophoresis on a 10% SDS polyacrylamide gel. Partitioned proteins were transferred to a nitrocellulose membrane. The membrane was probed with rabbit anti-Yap antibody (Cell Signaling Technology), rabbit anti-PABP antibody (Abcam), rabbit anti-eIF4G antibody (Cell Signaling Technology), mouse anti-eIF4A, anti-eIF4B or anti-eIF4E (Santa Cruz Biotechnology). HRP-conjugated anti-rabbit or anti-mouse IgG antibody (Jackson ImmunoResearch) was used as the secondary antibody. The corresponding protein bands were visualized using enhanced chemiluminescence reagents. The same membranes were re-probed with HRP conjugated GAPDH antibody (Proteintech), mouse anti-alpha tubulin antibody (Santa Cruz Biotechnology) or mouse anti-PCNA (Santa Cruz Biotechnology) to confirm equal loading of proteins for each sample.

At the end of the culture period, HK-2 cells were collected and lysed with RIPA buffer supplemented with complete protease inhibitor cocktail tablets. Protein concentrations were determined with the BCA Protein Assay Kit (Thermo Fisher Scientific), and protein lysates (30-50 μg) were mixed with (4x) NuPAGE LDS sample buffer, heated for 5 min at 99°C, and subjected to electrophoresis on 4%-15% polyacrylamide gradient SDS gels. The proteins were then transferred onto PVDF membranes (Millipore, Billerica, MA, USA) and incubated with antibodies against SGK1, phosphorylated-SGK1 (p-SGK1), GSK3β, phosphorylated-GSK3β (p-GSK3β), cleaved caspase-3, and COX IV (Cell Signaling Technology, Danvers, MA, USA); peroxisome proliferator-activator γ coactivator-1α (PGC-1α), Bcl-2, Bax, nuclear transcription factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM) (Abcam, Cambridge, UK); and Cytochrome C and GAPDH (Sigma-Aldrich, St. Louis, MO, USA). Immunoblots were developed using an HRP-conjugated anti-rabbit or anti-mouse IgG antibody (Jackson, West Grove, PA, USA). Relative concentrations were assessed by densitometry analysis of digitized autographic images using ImageJ software.