Anti synaptopodin

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.

The detailed immunofluorescent staining protocol and assessment of mitochondrial morphology were described previously^{11 (link)}. The following primary antibodies were used: anti-MRGPRD (Biorbyt, catalog no. orb157281, 1:100) and anti-synaptopodin (Santa Cruz Biotechnology, catalog no. sc-21537; 1:50). For mitochondria staining, a 200 nM solution of MitoTracker Red (ThermoFisher) was used. Images were taken on a confocal microscope (Eclipse Ti, Nikon Instruments). The average area, perimeter, axis length, and compactness of mitochondria were determined using CellProfiler software^{16 (link)}. There were 7–8 images taken into analyses per one condition, each image contained ~ 3000 segmented objects (mitochondria).

Recombinant ISM1 (endotoxin free) was produced by myBiosource (MBS1208159) expressed in yeast and purified near to homogeneity. Antibodies for immunofluorescence: anti-ISM1 was provided from Thermo Fischer (PA5-24968, 1:100 dilution); anti-β5 from R&D systems (AF8035, 1:20 dilution); anti-GRP78 from Genetex (GTX102567, 1:20 dilution); anti Cytochrome c from BD Pharmingen (556432, 1:400 dilution); anti-AIF from Santa Cruz (sc-13116, 1:200 dilution). Antibodies for immunoblotting and immunoprecipitation: anti-ISM came from Biolegend (622201, 1:500 dilution); anti-AIF from Sigma (A7549, 1:100 dilution); anti-cytochrome c from BD Pharmingen (556433, 1:100 dilution); anti-synaptopodin (sc-21537), anti-caspase-8 (sc-5263, 1:200 dilution); anti-caspase-3 (sc-7272, 1:200 dilution); anti-GRP78 (sc-166490, 1:100 dilution) and anti-αvβ5 (sc-13588, 1:100 dilution) were provided by Santacruz.