Fig 2: Identification of ARHGAP11A as a cell cycle regulated protein and a substrate of the APC/C.(A) MS and RNA-Seq quantitation for ARHGAP11A protein (left) and mRNA (right), respectively. (B) Immunoblot analysis of ARHGAP11A (HPA antibody) and GAPDH protein expression across asynchronous and elutriated NB4 cells. (C) Lysates from U2OS cells treated with either a non-targeting control siRNA (lane 1) or siRNAs targeting Cdh1 and Cdc20 (lane 2) were probed for levels of ARHGAP11A, Cdh1, Cdc20, cyclin B1, and GAPDH by immunoblot. (D) Asynchronous or serum-starved RPE-1 cells were treated with either a non-targeting control siRNA or an siRNA against Cdh1. Lysates were then probed with antibodies against ARHGAP11A (Bethyl antibody), Cdh1, cyclin B1, and GAPDH.DOI: http://dx.doi.org/10.7554/eLife.01630.017

Fig 3: Validation of anti-ARHGAP11A antibodies by siRNA-based depletion of ARHGAP11A protein.(A) HeLa cells were either mock treated, treated with siRNAs that target lamin A/C, or treated with different concentrations of siRNAs that target ARHGAP11A. Cells were cultured for 48 hr before harvest, lysis, and immunoblot analysis with a Human Protein Atlas (HPA) antibody recognizing ARHGAP11A (top) or an antibody recognizing lamin A/C (bottom). The anti-ARHGAP11A recognizes a band at the correct molecular weight (∼100 kDa), which is significantly decreased upon siRNA depletion of ARHGAP11A protein. Note that lamin A/C levels are not significantly perturbed by treatment of siRNA targeting ARHGAP11A. (B) U2OS cells were either mock treated or treated with siRNAs that target ARHGAP11A, incubated for 48 hr, lysed, and immunoblotted with antibodies recognizing ARHGAP11A (Bethyl, top) or alpha-tubulin (bottom). The anti-ARHGAP11A recognizes a band at the same molecular weight as the HPA antibody that is significantly decreased upon siRNA depletion of ARHGAP11A. Note that alpha-tubulin levels are unchanged +/− ARHGAP11A siRNA. *Non-specific bands.DOI: http://dx.doi.org/10.7554/eLife.01630.018