Sc 10815

BEAS-2B cells in each group were fixed using 4% polyformaldehyde for 30 min at room temperature (24°C). Cells were permeabilized using 0.1% Triton X-100 for 5 min at room temperature. Cells were subsequently blocked using 10% bovine serum albumin for 1 h at room temperature before incubation with anti-uPA (SC-59727, Santa Cruz, 1:125 dilution) and anti-uPAR (SC-10815, Santa Cruz, 1:200 dilution) overnight at 4°C. Cells were washed thrice using PBS and then incubated with antirabbit immunoglobulin G (IgG) for 1 h at 37°C and washed again thrice with PBS. Cell slides were finally stained with DAPI and mounted with an antifade mounting medium and imaged via confocal microscopy.

Cultured podocytes were lysed in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitors (Roche). The lysates were fractionated in SDS-PAGE, followed by protein transfer to blots and incubation with primary antibodies, including polyclonal anti-uPAR (1:200, SC-10815, Santa Cruz, Texas, USA), polyclonal anti-ITGB3 (1:200, SC-14009, Santa Cruz, Texas, USA), polyclonal anti-synaptopodin (1:200, SC-21536, Santa Cruz, Texas, USA), and polyclonal anti-GAPDH (1:5000, AP0063, Bioworld, MN, USA), respectively.