Anti tgfβ receptor 1 tgfβri

Protein lysates from heart samples were loaded on and separated by a 10–15% SDS-PAGE, and then transferred to PVDF membranes (Millipore, MA, United States). The membranes were probed with primary antibodies overnight at 4°C after blocking with 5% milk in Tris-buffered saline with 0.1% Tween (TBST). The primary antibodies were anti-TGFβ1, anti-TGFβ receptor I (TGFβRI), anti TGFβ receptor II (TGFβRII), anti-phosphorylated Smad2/3, anti-Smad2/3, and anti-Smad4 (Cell Signaling Technology, shanghai, China), anti-collagen I and III (Col I and III, Proteintech, Wuhan, China), anti-α-SMA, anti-periostin, anti-ACE (Abcam, Shanghai, China) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Multi Sciences, Hangzhou, China). Membranes were washed by TBST for three times and then incubated with appropriate secondary antibodies for 1 h at room temperature. After another three times wash by TBST, the signal was detected on FluorChemE (Protein Simple, CA, United States). The densities of bands were quantified by an ImageJ Analysis System and expressed as ratios to GAPDH.

After 48 h of treatment with different concentrations of PD, HSFs were lysed in radioimmunoprecipitation assay (RIPA) buffer for 30 min on ice. After lysis was complete, the liquid was collected and centrifuged for 10 min. The supernatant was used to measure the protein concentration (BCA protein assay kit, Thermo Fisher Scientific, USA). Extracted protein (20 mg) was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After being blocked with 5% bovine serum albumin (BSA), the membrane was incubated overnight at 4 °C with primary antibodies. The primary antibodies were anti-type I collagen (Col I), anti-α-SMA, anti-matrix metalloproteinase 2 (MMP2), anti-type III collagen (Col III), anti-TGF-β1, anti-TGF-β receptor I (TGFβ RI), anti-phospho-Smad2/3 (p-Smad2/3) and anti-Smad2/3 (all from Cell Signaling Technology, USA). The next day, the PVDF membrane was incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA) on a slow shaker at room temperature for 1 h. Then, the membrane was washed 3 times and analyzed with Odyssey V3.0 image scanning (Li-COR. Inc., Lincoln, NE, USA). β-actin (Cell Signaling Technology, USA) was used as a loading control.