Rabbit polyclonal anti phospho cdk2

Immunoblotting was performed as decribed in51 (link). Primary antibodies used were mouse monoclonal anti-CDK2 (Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit polyclonal anti-CDK2 (Abcam, Cambridge, UK), rabbit polyclonal anti-phospho-CDK2 (Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-cyclin E2 (Cell Signaling Technology), rabbit polyclonal anti-CDK4 (Cell Signaling Technology), rabbit polyclonal anti-p38 MAPK, rabbit polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182), rabbit polyclonal anti-phopho-Akt (Ser473), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology), mouse monoclonal anti-Pan Akt (R&D Systems, Minneapolis, MN), rabbit polyclonal anti-Bax and rabbit polyclonal anti-Bcl2 (Abcam) and mouse monoclonal anti-tubulin IgGs (Sigma-Aldrich). Alexa Fluor 680 (Invitrogen) and IRDye 800 (LI-COR, Lincoln, NE) donkey anti-rabbit, anti-goat or anti-mouse IgGs were used as secondary antibodies. Detection and quantification was performed with an Odyssey Infrared Imager (LI-COR). Full Western blots with antibodies against CDK2 and phospho-CDK2 are shown in Supplementary Figure S5.

Podocytes cultured on black 96-well plates (PerkinElmer, Waltham, MA, USA) were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS and permeabilized with 0.1% Triton X-100 in PBS. Cells were incubated with rabbit anti-cleaved caspase-3 (Cell Signaling Technology), rabbit polyclonal anti-phospho-CDK2 (Cell Signaling Technology), or rabbit anti-CDK2 (Abcam) at room temperature for one hour, followed by IRDye 800 (LI-COR) donkey anti-rabbit IgG and 1 μM DRAQ5^TM (Thermo Fisher Scientific, Waltham, MA, USA) at RT for one hour. Detection and quantification were performed with an Odyssey Infrared Imager (LI-COR). DRAQ5^TM was used for normalization.