Thrombospondin 1

After 48 hours of transfection/overexpression, cells were collected and lysed with cell lysing buffer containing protease inhibitors on ice. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4 °C, and the supernatant was collected. After determining the protein concentration, the sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a Poly vinylidene fluoride (PVDF) membrane. The membrane blocked with 5% skimmed milk for 1 hour. Then, the membrane was incubated with primary antibodies (mouse anti-human) of Act B, endothelin-1 (ET-1), thrombospondin-1 (Tsp-1), TGF-β1, Oncostatin M (OSM), and Smad2/3 (Santa Cruz, CA) overnight at 4 °C. After washing, the membrane was incubated with the secondary antibody at room temperature for 1 hour. The membrane was finally colored with chemiluminescence reagent. The protein bands were visualized in a gel imager (Bio-Rad Laboratories Inc., Hercules, CA).

Normal human fibroblasts or RDEB fibroblasts were plated on coverslips (Thomas Scientific, 6661-K40) in 6-well plates (Thermo Scientific, Catlog No. 140675) at 0.5 × 10⁵ cell/well. After 48 hours, cells were fixed by 4% paraformaldehyde and blocked by 3% fetal bovine serum in 0.1% Triton X-100 for half hour at room temperature. Primary antibodies used were Collagen VII (Sigma Prestige, HPA042420, Rabbit, 1:50 dilution), Thrombospondin-1 (Santa Cruz, sc-59887, mouse, 1:50 dilution), SEC23 (Abcam, ab99552, goat, 1:100 dilution), SEC31 (Santa Cruz, sc-376587, mouse, 1:50 dilution), pSMAD3 S423/S425(Rockland, 600-401-919, Rabbit, 1:50 dilution), Calreticulin (Life Span Bioscience, LS-B5223-125, Sheep, 1:50 dilution) and Collagen I (SouthernBiotech, 1310-01, goat, 1:50). Cells with primary antibodies were incubated for 2 hours at room temperature. Secondary antibodies, Alexa Fluor 594 goat anti-rabbit (1:800) (Invitrogen, Eugene, OR), Alexa Fluor 488 goat anti-mouse (1:250) (Invitrogen, Eugene, OR), Alexa Fluor 647 donkey anti-goat (Invitrogen, Eugene, OR) and Alexa Fluor 488 donkey anti-sheep (1:250) (Invitrogen, Eugene, OR), were applied for 1 hour at room temperature. Coverslips were mounted on the slides with DAPI Fluoromount-G (SouthernBiotech, #0100-20) and analyzed by confocal microscopy (Nikon A1R Microscope).