Hrp conjugated secondary antibody

Cells and ventricular tissue were lysed with RIPA buffer (Sigma–Aldrich, USA), and the protein concentrations of the lysates were determined using BCA Protein Assay Reagent (Pierce Biotechnology, USA). Total protein samples (40 μg) were separated electrophoretically, transferred to a PVDF membrane and probed using monoclonal primary antibodies against PI3K (1:5000, Abcam, ab139307), p-PI3K (1:1000, Abcam, ab32089), AKT (1:1000, Abcam, ab179463), p-AKT (1:1000, Cell signaling, 4060s), KDM5A (1:5000, Abcam, ab194286), IGF1 (1:1000, Abcam, ab223567), MYH11 (1:1000, Abcam, ab82541), TGFB3 (1:1000, Abcam, ab15537), and β-actin (1:500, Abcam, ab5694), followed by an HRP-conjugated secondary antibody (1:5000; Abbiotec, USA). Bands were exposed using an ECL kit (Bio–Rad, USA) and analyzed using Image-Pro Plus software. LY294002 (20 μmol/L, Cell signaling, 9901s) is an inhibitor of PI3K, and ARQ-092 (10 μmol/L, Abcam, ab235550) is an inhibitor of AKT.

Immunohistochemical staining was conducted to investigate the expression of BTLA in kidney graft sections as previously described^{42 (link)}. The primary BTLA antibody (Abbiotec CA, USA) along with HRP-conjugated secondary antibody was used. Eight images under a high power field (200×) were captured randomly from each specimen in each group using Image Pro Plus 5.0 software (Media Cybernetics MD, USA), with the integrated optical density (IOD) value to express the relative quantity of BTLA. Images were captured using a Nikon Eclipse 50i microscope (Nikon, Japan) along with Imaging Software NIS-Elements (Nikon, Japan).
For immunofluorescence staining, frozen tissue sections of kidney grafts were blocked with goat serum for 30 min at room temperature and then incubated with anti-rat CD4 and anti-rat CD8 primary antibodies (Santa Cruz CA, USA) at 4 °C overnight and with FITC goat anti-rat IgG at 37 °C for 1 h.