Supersignal west dura extended duration substrate

Cultured macrophages were lysed on ice with cold radio immunoprecipitation assay (RIPA) lysis buffer (in house) supplemented with protease inhibitors (Complete protease inhibitor cocktail tablets, Roche Diagnostics, #11697498001) and protein concentration was measured using BCA protein assay (Pierce BCA Protein Assay Kit, #23225, ThermoFisher Scientific). Equal amounts of protein were loaded onto 10% SDS-polyacrylamide gel and transferred to an Immobilon-P transfer membrane (# IPVH00010, PVDF membrane, Millipore). The blots were blocked for 1 h using 10% milk in Tris-Buffered Saline and 0.1% Tween-20 solution and incubated O/N with goat anti-mouse endoglin (1:1000 dilution, BAF1097, R&D Systems) or mouse anti-β-Actin (1:10.000 dilution, A5441, Sigma-Aldrich). Blots were incubated for 1 h with horse radish peroxidase anti-goat (goat anti-mouse IgG Poly-HRP Secondary Antibody HRP conjugate, #32230, ThermoFisher Scientific) or anti-mouse (ECL mouse IgG, HRP-linked whole Ab #NA931, Sigma-Aldrich, GE Healthcare, UK). Blots were developed in a Kodak X-omat 1000 processor with Thermo Scientific SuperSignal West Dura (Extended Duration Substrate) or SuperSignal West Pico and exposed to Fuji SuperRX medical X-ray film. Analysis was performed using Image J (National Institute of Mental Health, Bethesda, Maryland, USA).

For Western blots, initial processing of PVDF membranes followed a similar protocol as that described above for detection of bound fluorescent peptides to PVDF protein blots. However, after blocking with casein/TBST (ThermoFisher, Inc) for 4 h, PVDF membranes were incubated overnight at 4 °C with primary antibody diluted in 1× casein/TBST (ThermoFisher, Inc). Primary antibody was removed with multiple washings, and blots were then incubated with horseradish peroxidase–conjugated secondary antibody diluted in 1× casein/TBST for 2 h at 4 °C. Blots were finally washed multiple times to remove free secondary antibody and then incubated with ThermoFisher SuperSignal West Dura extended duration substrate and then imaged in a Fuji ImageQuant LAS 4000 Imager.