Rabbit anti pdgfrα antibody

Total proteins were lysed by RIPA buffer (Beyotime, China). After centrifugation (4°C, 12,000 g for five minutes), protein concentrations were measured by Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, CA, USA). SDS-PAGE was conducted to separate equal amounts of proteins, which were blotted onto PVDF membrane (Millipore, USA). After blocked with milk, the membranes were incubated overnight at 4°C with primary antibodies. Primary antibodies were as follows: rabbit anti-p-c-Kit (Tyr719) antibody, rabbit anti-c-Kit antibody (CST, USA); rabbit anti-α-SMA antibody, rabbit anti-p-PDGFRα (Tyr720) antibody, rabbit anti-PDGFRα antibody (Abcam, USA); and mouse anti-GAPDH antibody (Proteintech, USA). Afterwards, membranes were incubated with the second antibody (Invitrogen, USA) at 25 °C for one hour. Finally, the blots were detected with Kodak film developer (Fujifilm, Japan). The protein blots were analyzed by the ImageJ software.

Mouse heart was perfused with PBS for 3 min followed by 4% paraformaldehyde in PBS for 3 min for fixation. After perfusion, heart was removed from mice and immersed in 4% paraformaldehyde in PBS for 2 h at 4°C to complete fixation. After fixation, the heart was placed in 30% sucrose in PBS overnight or until tissue sinks at 4°C before embedding in OCT. Cryostat sectioning was performed to obtain heart sections in a 100 μm-thickness. Sections were washed in PBS, permeabilized in 0.5% Triton X-100 in PBS and blocked in 10% normal goat serum and 0.3% Triton X-100 in PBS. After blocking, sections were incubated with Rabbit anti-PDGFRα antibody (Abcam). Goat anti-Rabbit IgG conjugated with Alexa Fluor 555 (Thermo Fisher) was used as secondary antibody and DAPI was used for counterstaining. Images were acquired using Zeiss LSM 700 microscope (Zeiss) at 20X and 40X magnification with the ZEN Black software (Zeiss). ImageJ/Fiji was used to process images.

Cell surface proteins were labeled with biotin by incubation of cells in 1× PBS containing 0.25 mg/ml EZ-Link Sulfo-NHS-SS-Biotin for 30 min on ice. To detect localization of PDGFRα specifically at the cell surface upon ligand addition, cells were stimulated with 50 ng/ml PDGF-AA for the indicated times before biotin labeling. To detect internalized PDGFRα, cells were stimulated after labeling with biotin, and biotinylation of proteins at the cell surface was removed by treatment with glutathione-stripping solution (50 mM reduced glutathione, 150 mM NaCl, 70 mM NaOH, 1.25 mM MgSO₄, 1.25 mM CaCl₂, and 1 mM EDTA, pH 8.5) for 10 min on ice. For total amounts of biotinylated PDGFRα receptor, the same procedure was applied, except for stripping of biotin from cell surface proteins. SDS-PAGE and WB analyses were performed with the rabbit anti–PDGFRα antibody (Abcam).