Essentially fatty acid free bsa

Subconfluent MRC5 cells were plated at 200000 cells/well (six well plates), starved for 24 hours in the presence of 0.05% essentially fatty acid free BSA (Sigma) and treated with or without TGFβ (4 ng/ml) during 24 or 48 hours. Cells were washed with phosphate-buffered saline (PBS) and then lysed as described [30] (link). Equal amounts of total protein extracts were analyzed by SDS-PAGE, then transferred on a polyvinylidene difluoride membrane (PVDF) and subjected to antibodies against PDGF-D (1 µg/ml, Santa Cruz), PDGF-C (1 µg/ml, monoclonal antibody) or β-actin (0.5 µg/ml, Sigma).

Recombinant proteins or peptides were diluted to 10 µg/mL in PBS, and 25 µL of the dilutions per well was used for coating at 4°C overnight in 384-well microtiter plates with high-binding surface (Corning, Tewksbury, MA, USA). The wells were then incubated with blocking solution (2% essentially fatty acid-free BSA (Sigma-Aldrich) in PBS at room temperature for 1.5 h, washed with PBS for 30 sec, and incubated at room temperature for 1.5 h with different concentrations of recombinant proteins in the presence of different concentrations of peptides. After washing 3 times with PBST (PBS with 0.05 % Tween 20) for 30 s, primary antibodies (diluted 1:300 in PBS) were applied for 1 h. The ELISA plate was washed 3 times for 5 min with PBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (diluted 1:1000 in PBS). The plate was rinsed again 3 times with PBST for 5 min. As horseradish peroxidase substrate, 25 µL ortho-phenylenediamine dihydrochloride (Thermo Fisher Scientific; 1 mg/mL) was added per well, and plates were incubated for 0.5–5 min. The color reaction was terminated with 2.4 M sulfuric acid, and the absorption was measured at 492 nm using the μQuantTM microplate spectrophotometer (Bio-Tek Instruments, Bad Friedrichshall, Germany).