Anti fcrn

Cells were washed with PBS and lysed in Laemmli buffer (Invitrogen, USA) containing 50 mM DTT. Lysates from 2x10⁵ cells were analysed in singlicate by 15% SDS-PAGE, and proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in PBS and incubated with the antibodies anti-FcRn (Santa Cruz Biotechnology, USA sc-271745), anti-ATG5 (Cell Signaling Technology, USA, #12994) and anti-LC3-I/II (Cell Signaling Technology, #4108S). Anti-β-actin (Santa Cruz Biotechnology, #sc-47778) or anti-GAPDH (Santa Cruz Biotechnology, # sc-365062) was a loading control. The membranes were developed with an enhanced chemiluminescence Western blot detection reagent (Thermo Scientific, USA). Uncropped Western blot and replicates are presented as Supplemental Figure S1 and S2 respectively. Densitometry of the bands was quantified by using ImageJ. FcRn, ATG-5 and LC3-I/II expression was normalized to β-actin or GAPDH expression.

Western blotting was performed according to the protocol of Bio-Rad semi-dry transfer using the Bio-Rad Transfer Cell System. Podocytes were harvested by scraping into RIPA buffer and protein concentrations were determined by bicinchoninic acid assay (Thermo Fisher Scientific Cat# 23225). Anti-FcRn (1:1,000, Santa Cruz, Cat# 271745) was used as primary antibody in detection. Goat anti-mouse IgG-HRP (1:5,000, Santa Cruz, Cat# 2005)was used as secondary. The antibody complexes were detected using enhanced chemiluminescence (Tanon, Cat# 180501) and Western blot images were captured using a photodocumen-tation system (Tanon-4500).