Baf4910

Podocyte and tubular cell isolation from mouse kidneys were performed as previously described with slight modifications^{51 (link),52 (link)}. In brief, podocytes were ex vivo selected by biotin-labeled anti-Kirrel3 and podocalyxin antibodies (R&D Systems BAF4910 and R&D Systems BAF1556, respectively), then isolated using magnetic, streptavidin labeled Dynabeads (Thermo Fisher). To isolate tubular cells, dissected and minced kidneys were digested with collagenase type II in RPMI media for 30 mins at 37°C. Cells were sieved first through a 100 μm nylon mesh, then through a 40 μm nylon mesh, followed by centrifugation at 500 g for 10 min. The pellet was resuspended in red blood cell lysis buffer (R&D Systems) and incubated on ice for 10min. After centrifuge at 500 g for 10 min, cells were resuspended in RIPA buffer (TEKnova) containing 1% protease inhibitor cocktail (Sigma) and stored at −80°C freezer for experiments.

Podocyte and tubular cell isolation from mouse kidneys were performed as previously described with slight modifications^{50 (link),51 (link)}. In brief, podocytes were ex vivo selected by biotin-labeled anti-Kirrel3 and podocalyxin antibodies (R&D Systems BAF4910 and R&D Systems BAF1556, respectively), then further purified using magnetic, streptavidin conjugated Dynabeads (Thermo Fisher). To isolate tubular cells, dissected and minced kidneys were digested with collagenase type II in RPMI media for 30 min at 37 °C. Cells were sieved first through a 100 μm nylon mesh, then through a 40 μm nylon mesh, followed by centrifugation at 500 g for 10 min. The pellet was resuspended in red blood cell lysis buffer (R&D Systems) and incubated on ice for 10 min. After centrifuge at 500 g for 10 min, cells were resuspended in RIPA buffer (TEKnova) containing 1% protease inhibitor cocktail (Sigma) and stored at -80^oC freezer for experiments.