Gapdh specific antibody

Cultured or transfected cells were lysed in RIPA buffer with 1% PMSF. Western blot was performed on 10% SDS-PAGE using Mini-PROTEAN^® Tetra Cell Systems (Bio-Rad). Proteins were transferred onto polyvinylidine difluoride (PVDF) membranes (Immobilon, Millipore). Membranes were incubated with anti-PRB4 rabbit polyclonal antibody (Abcam), anti-NSD1 rabbit monoclonal antibody (Abcam) or PI3K rabbit monoclonal antibody (Cell Signaling) or Akt mouse monoclonal antibody (Cell Signaling) or phospho-Akt rabbit monoclonal antibody (Cell Signaling) or Bad Rabbit monoclonal antibody (Cell Signaling) at 1:1000 dilution, or GAPDH specific antibody (Sigma-Aldrich) at 1:5000 dilution at 4°C overnight. Signals were visualized using ECL Substrates (Millipore, MA, USA).

Renal tissue from the corticomedullary area or HK-2 cells was lysed and denatured at 100°C for 5 min in a SDS buffer and separated by 8%, 10% or 12% PAGE gels. The proteins were transferred on to a PVDF membrane, blocked for 1 h with 5% (w/v) dried non-fat skimmed milk powder in TBST (TBS containing 0.1% Tween 20) and probed with the indicated antibodies. The following primary antibodies were used: rabbit antibodies against YAP, pYAP, Mst1, pMst1, vimentin (Cell Signaling Technology, 1:1000 dilution), αSMA (Abcam, 1:1000 dilution), AQP1 (Millipore, 1:1000 dilution) and CTGF (connective tissue growth factor) (Santa Cruz Biotechnology, 1:400 dilution); and mouse antibodies against PCNA (Cell Signaling Technology, 1:2000 dilution) and Na⁺/K⁺-ATPase (DSHB, 1:1000 dilution). Horseradish peroxidase-conjugated secondary antibodies were applied, and ECL (Pierce) was conducted to detect proteins. A GAPDH-specific antibody (Sigma) was used for loading controls on stripped membranes. Quantification of immunoblots compared the relative ratio of the gray value of pYAP, YAP and pYAP/YAP at all time points with the controls. The results were measured using ImageJ software (NIH) and normalized to GAPDH.