Anti α smooth muscle actin α sma

NRK-52E cells were cultured with TGF-β1, AA, and/or SSBE in 6-well plates at a seeding density of 3×10⁵ cells/well containing glass slides. Cells were washed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (Sigma-Aldrich) at 4°C for 30 min. Following permeabilization with 0.1% Triton X-100 for 10 min, specimens were washed with PBS, and the substrate was blocked with 10% FBS to eliminate nonspecific fluorescence. Immunofluorescence staining was performed using anti-Col3α1 (dilution 1:200), anti-E-cadherin (cat. no. ab53033, dilution 1:400; Abcam, Cambridge, MA, USA), and anti-α-smooth muscle actin (α-SMA; dilution 1:400; cat. no. sc-32251), anti-protein patched homolog 1 (Ptch1; dilution 1:400; cat. no. sc-9016), anti-smoothened (Smo; dilution 1:400; cat. no. sc-13943) and anti-Gli family zinc finger 1 (Gli1; dilution 1:100; sc-6153), purchased from Santa Cruz Biotechnology, primary antibodies at 4°C overnight. Following washing with PBS three times, the cell preparations were incubated with fluorescein isothiocyanate (green)/tetramethylrhodamine-(red) labeled secondary antibodies (dilution 1:2,000; Sigma-Aldrich) for 1 h at room temperature. Following washing with PBS, cell preparations were placed in acacia and covered with a slide. Immunofluorescence studies were semi-quantitatively or quantitatively assessed by two blind independent investigators.

As described by Takikita-Suzuki et al[20 (link)], the frozen kidney tissue was homogenized on ice in buffer containing Tris-HCl (20 mmol/L; pH 7.5), ethylenediamine tetraacetic acid (1 mmol/L), NaCl (140 mmol/L), Nonidet P-40 (1%), aprotinin (50 μg/mL), NaF (50 mmol/L), sodium orthovanadate (1 mmol/L) and phenylmethyl sulfonyl fluoride (1 mmol/L). Homogenized tissue was centrifuged for 30 min at 15000 rpm at 4 °C. Supernatant was collected and protein concentrations were measured using the method of Bradford. After 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was transferred to a nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, England, United Kingdom). Blocking was performed for 2 h in phosphate-buffered saline (PBS) solution containing 5% (w/v) skim milk, followed with incubation with anti-α smooth muscle actin (α-SMA; Abcam) or anti-pPDGFRβ (Antibody #3161, Cell Signaling) antibodies, followed by an appropriate HRP-conjugated secondary antibody (Cell Signaling). Proteins were detected using an enhanced chemiluminescence kit (Amersham/GE Healthcare, United Kingdom). Densitometric analysis for pPDGFRβ was performed with normalization to GAPDH.