Odyssey v3.0 image scanning

Western blot analyses were performed as previously described^{38 (link)}. Various antibodies, including rabbit monoclonal anti-REST (Abcam, Cambridge, UK), mouse anti-nestin (BD, San Jose, CA, USA), rabbit monoclonal anti-Pax-6 (Biolegend, San Diego, CA, USA), mouse monoclonal anti-β3-tubulin (Millipore, Billerica, MA, USA), mouse monoclonal anti-rhodopsin, rabbit polyclonal anti-recoverin (Millipore), mouse monoclonal anti-Brn3a (Millipore) and mouse monoclonal anti-Caspase-3 (Santa Cruz, California, USA) were diluted 1:1000, and mouse anti-β-actin (Sigma-Aldrich) was diluted 1:5000. The membranes were incubated with 1:5000 dilutions of DyLightTM680-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Sigma-Aldrich), and analyzed by Odyssey V 3.0 image scanning (LI-COR, Lincoln, NE, USA).

Cells were washed with ice-cold PBS (6 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.4, 140 mM NaCl, 3 mM KCl) and lysed in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% Na Deoxycholate, 1 mM Orthovanadate, 5 mM NaF, 2.5 mM Na₄P₂O₇) and cOmplete Protease Inhibitor Cocktail (catalog 12352200, Roche) for 20 minutes on ice. The cell lysates were centrifuged (8000g) for 10 minutes. Proteins (10–20 μg) were resolved on SDS-PAGE under reducing conditions on a 10% polyacrylamide gel. The Western blot system was established using the Bio-Rad Bis-Tris Gel system according to the manufacturer’s instructions. Primary antibodies were prepared in 5% BSA blocking buffer at a dilution of 1:1000 and incubated with the nitrocellulose membrane (Merck Millipore) at 4°C overnight, followed by washing and incubating with HRP-conjugated secondary antibodies for 1 hour at room temperature. The immunoblots were visualized by Odyssey V3.0 image scanning (LI-COR). The intensities of the bands were quantified using NIH ImageJ software, and the values were normalized to the β-actin or GAPDH values for each sample. And the relative protein levels were expressed as the fold change relative to the Ctls.

Cells were lysed with RIPA buffer (Beyotime, Shanghai, China), then the lysate was centrifuged at 12,000 rpm for 10 min, and the protein in the supernatants was collected and quantified. Each protein lysate (30 µg) was resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Following transfer, membranes were blocked with 5% skim milk for 2 h and probed with primary antibodies at 4 °C overnight and incubated with appropriate secondary antibodies. Antibody reactivity was detected by exposure in an Odyssey V3.0 image scanning (Li-COR Inc., Lincoln, NE, USA).

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.