Anti pdgfrα

HEK293T cells were seeded (7.5 × 10⁶ cells) into 90 mm dishes and transfected with wild-type or mutated PDGFRα expression vectors using Lipofectamine 3000. Forty-eight h after, the transfected cells were lysed using the Mem-PER Plus membrane protein extraction kit (Thermo Fisher Scientific), and proteins were visualized via fluorescent western blotting. Briefly, proteins were resolved by SDS-PAGE and transferred onto PVDF membranes. Next, membranes were blocked and incubated with antibodies using the protocol described above. The anti-PDGFRα (#5214, Cell Signaling Technology) and anti-phosphotyrosine (4G10) antibodies were used as the primary antibodies, and IRDye 680RD donkey anti-rabbit IgG and IRDye 800CW donkey anti-mouse IgG (LI-COR, Lincoln, NE, USA) were used as the secondary antibodies. Fluorescence detection and quantification were performed using Odyssey CLx (LI-COR).

After the reperfusion, fresh heart biopsies were fixed in 4% paraformaldehyde overnight at 4°C and embedded in paraffin. Sections into 5-μm slices were stained with hematoxylin-eosin (H&E) or Masson’s trichrome for assessment of fibrosis. Tissues for immunofluorescence were submerged in liquid nitrogen and then embedded in optimal cutting temperature (OCT) solution (Sakura Finetek, USA) on dry ice to be frozen completely. Cardiomyocyte hypertrophy was examined in the peri-infarct zone. Myocyte cross-sectional areas were measured using Image J software (National Institutes of Health) in frozen sections stained with 5 μg/ml wheat germ agglutinin (WGA-Alexa Fluor^® 488 conjugate, Invitrogen, USA). Five parts were chosen in the WGA images (200X) including left top, right top, middle, left bottom, and right bottom, and six cells were analyzed for each part. In other experiments, the hearts were excised for Masson staining to evaluate the cardiac remodeling. For inflammatory cell infiltration and PDGFR protein expression, immunofluorescence staining with anti-CD45 (Cell Signaling Technology, USA) and anti-PDGFRα (Cell Signaling Technology, USA) in frozen sections was conducted. Then the number of CD45+ cells/field were quantified by Image J software (National Institutes of Health, USA).

Tissue or cells were homogenized in lysis buffer containing 50 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 1% IGEPAL CA-630, 0.1% SDS, 0.5% deoxycholic acid, 1 mmol/L EDTA, 0.1 mmol/L Na₃VO₄, 1 mmol/L NaF, 50 μmol/L phenylmethylsulfonyl fluoride (PMSF), 5 μg/mL aprotinin, and 5 μg/mL leupeptin. Following SDS-PAGE, immunoblotting was performed using the following antibodies: anti-YAP (Cell Signaling #14074, 1:1000), anti-cardiac troponin T (Thermo Fisher MA5-12960, 1:2000), anti-GFP (Cell Signaling #2956, 1:1000), anti-GAPDH (Cell Signaling #5174, 1:1000), anti-PDGFRα (Cell Signaling #3174, 1:1000), anti-Hsp47 (Enzo Life Sciences #ADI-SPA-470, 1:1000), and anti-tubulin (Sigma, T-6199, 1:1000). Densitometry was performed using NIH ImageJ software.