Chemiluminescent peroxidase substrate

Proteins from the renal cortex or HUVECs were extracted in RIPA buffer with proteinase inhibitors, and protein concentrations were determined using the BCA assay. Twenty-five microgram of proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with Roti-block (Roth, Karlsruhe, Germany) for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C and corresponding secondary antibodies for 1 h at room temperature. The proteins were visualized using a chemiluminescent peroxidase substrate (Roche, Mannheim, Germany; or Thermo Scientific, Rockford, USA). Protein expression was quantified using Image J (NIH, USA). Specific primary antibodies used: mouse-anti-α-SMA (Sigma-Aldrich, A5228, 1:2,000), goat-anti-CTGF (Santa Cruz, sc-14939, 1: 200), mouse-anti-γ-tubulin (Sigma, T6557, 1:10,000). The secondary antibodies conjugated with horseradish peroxidase: rabbit-anti-goat (Sigma-Aldrich, A8919), horse-anti-mouse (Cell Signaling, 7076, 1:20,000).

The cell lysates of hiPSC-CMs were used for protein isolation. Western blotting was performed using proteins extracted with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 1% Triton X-100, 1% sodium deoxycholate). The proteins were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes. After blocking with Roti-block (Roth, Karlsruhe, Germany), membranes were incubated with primary antibodies overnight. Immunocomplexes were incubated with corresponding secondary antibodies and visualized using a chemiluminescent peroxidase substrate (Roche, Mannheim, Germany; or Thermo Scientific, Rockford, IL, USA). Protein expression was quantified using Image J (NIH, Bethesda, MD, USA). Specific primary antibodies used were mouse-anti-NDPK-B (MC-412; Kamiya, Seattle, WA, USA), mouse-anti-γ-tubulin (Sigma-Aldrich) and mouse-anti-SK4 (sc-365265, Santa Cruz Biotechnology, Heidelberg, Germany).The corresponding secondary antibody was rabbit anti-mouse peroxidase (Sigma-Aldrich).