Fig 1: RSL24D1 and WDR12 associate with RPA194 and, along with BOP1 and PES1, regulate its levels. (A) MCF10A whole cell extracts were immunoprecipitated with α-RPA194 antibody with/without RNase A treatment. Input corresponds to 0.75% of the whole cell extract (WCE) used for immunoprecipitation. Unconjugated protein A beads were used as a negative control. RPA194, PES1, WDR12, and RSL24D1 were detected by western blotting with specific antibodies. Representative western blot images shown are one of two biological replicates. (B) Panel as above, except MCF10A whole cell extracts were immunoprecipitated with α-WDR12 antibodies. (C) α-RPA194, α-WDR12 antibodies for immunoprecipitation and α-PES1 antibody for blotting are specific. Western blot of MCF10A cell lysate using α-RPA194 (left), α-PES1 (middle), and α-WDR12 (right) antibodies. α-β-actin was used as an internal loading control. Mock and nontargeting (siNT) were used as negative controls, siRPA194, siPES1, and siWDR12 were used as positive controls to confirm a reduction in levels of their respective expected bands. (D) Depletion of RSL24D1 and PeBoW reduce RPA194 protein levels in MCF10A cells. Cells were treated with the indicated siRNAs for 72 h and total protein from whole cell extracts was harvested. Mock, nontargeting (siNT), and SBDS siRNAs are shown as negative controls. (Left) Representative western blot images using α-RSL24D1 and α-β-actin antibodies. (Right) Quantitation of RPA194 levels is reported relative to siNT and normalized to the β-actin loading control. Graph indicates mean ± SD, n = 3 biological replicates. Data were analyzed by one-way ANOVA with Dunnett's multiple comparisons test where (****) P ≤ 0.0001; (**) P ≤ 0.01; ns = not significant.