Phospho smad3

Each group of HaCaT cells was washed with PBS followed by fixation with 4% paraformaldehyde (pH 7.4) in 6-well plates. Cells were incubated with 0.5% Triton X-100 for 30 min at room temperature, and treated with 5% BSA for 1 h. Cells were incubated with the following primary antibodies: Smad3 (#9523; dilution, 1:100), phospho-Stat3 (#9145; dilution, 1:100), Stat3 (#12640; dilution, 1:500) (all from Cell Signaling Technology), and phospho-Smad3 (ab52903; dilution, 1:100; Abcam) overnight at 4°C. The cells were incubated with the corresponding fluorescent dye-conjugated secondary antibodies (#4412; dilution, 1:200; Cell Signaling Technology) at 37°C for 1 h, and protected from light. The cells were visualized via fluorescence microscopy.

Tissue protein extracts were generated using RIPA buffer (150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM NaF, 50 mM Tris pH 7.4) with protease (cOmplete tablets, EDTA free, Roche) and phosphatase (phosphatase inhibitor cocktail sets I + II, Millipore) inhibitors. Standard Western blotting analysis was performed using 10% SDS-PAGE gels with the following primary antibodies from Cell Signaling: SMAD3 (1:1000, #9523), Phospho-SMAD3 (1:1000, #9520), SMAD2 (1:1000, #5339), Phospho-SMAD2 (1:1000, #3108), ERK1/2 (1:1000, #4695), Phospho-ERK1/2 (1:2000, #4370), p38 (1:1000, #8690), and Phospho-p38 (1:1000, #4511), and the following antibody from Abcam: Total OXPHOS Rodent WB Antibody Cocktail (1:5000, ab110413). Secondary antibody incubations were performed at room temperature for 60 min using HRP-conjugated antibodies (1:10,000) and then imaged using a ChemiDoc system (Bio-Rad).
Quantification of the TGF-β1 content was performed on cardiac tissue and plasma using an ELISA kit by R&D Systems (DB100B) according to the manufacturer's instructions. The total TGF-β1 amounts were measured by first activating the samples (to convert biologically inactive latent TGF-β1 into active TGF-β1) as instructed by the kit protocol, while measurement of the “bioavailable” TGF-β1 content was performed by skipping the initial activation step.

Total cellular protein was extracted in radioimmunoprecipitation assay lysis buffer (1% Nonidet P‐40, 0.1% SDS, 1 mmol/l phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 1 mmol/l sodium orthovanadate and 1 mmol/l sodium fluoride in phosphate‐buffered saline), and the protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). From each sample, 50 µg of protein were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Boston, MA, USA). Membranes were incubated with primary antibodies (rabbit monoclonal antibodies against human α‐SMA, phospho‐Smad3 and TGF‐β1, all obtained from Abcam, Cambridge, UK) at 4 °C overnight and secondary antibodies (goat anti‐rabbit IgG, Abcam) for 1 h at room temperature. The specific protein was detected using an enhanced chemiluminescence system (Amersham, Arlington Heights, IL, USA). β‐Actin was used as the loading control in all cases.