Fig 2: Cardiac expression of PP2A catalytic, scaffolding and regulatory subunits. Protein levels of PP2AC a isoform, PP2AC a and ß isoforms, PP2AA a and ß isoforms, B55a, B56?, B56d, and a4 in ventricular tissue from WT, HET, and HOM mice, assessed by immunoblotting. Lines show mean ± SE (n = 5–6 per group). There were no significant differences between groups (one-way ANOVA).

Fig 3: Characterization of adenoviral vectors. ARVM transduced with AdV.GFP, AdV.GFP-B56d-WT or AdV.GFP-B56d-S573A were maintained in culture for 18 h. In C, D and E, cells were exposed to vehicle (VEH) or isoprenaline (ISO). A. Expression of PP2AA, PP2AC, B55a, B56a and B56? in transduced cells. Quantitative data (mean ± SEM) show endogenous PP2A subunit expression normalized to the expression of calsequestrin. *P < 0.05 vs GFP (n = 3). B. GFP, GFP-B56d-WT and GFP-B56d-S573A were immunoprecipitated from lysates of transduced cells with a GFP antibody. GFP-B56d-WT, GFP-B56d-S573A, GFP, PP2AA and PP2AC were detected by immunoblot analysis. C. Phosphorylation of S573 in heterologously expressed B56d. ISO-induced phosphorylation of S573 in heterologous (GFP-B56d) and endogenous B56d is indicated. D. Cells lysed in buffer containing 1% Triton-X100 were subjected to subcellular fractionation. Immunoblots show detection of GAPDH, cTnI, GFP-B56d-WT and GFP-B56d-S573A in soluble (SOL) and insoluble (INSOL) subcellular fractions. The distribution of GFP-B56d-WT phosphorylated at S573 is also shown. E. Protein phosphatase activity was measured in whole cell lysates using pNPP as substrate. The specific activity of PP2A was determined by using 5 nM okadaic acid. Quantitative data (mean ± SEM) show the ISO-induced change in PP2A activity. In each experiment, the change in PP2A activity was expressed as a percentage of the basal activity. *P < 0.05 vs GFP, ?P < 0.05 vs SA (n = 6).

Fig 4: BOD1L1 interacts with the PP2A-B56 phosphatase complexA) Cartoon illustration depicting the experimental scheme used to discover protein interactors of BOD1L1. SW620 cells were first synchronized by thymidine-nocodazole. BOD1L1 was then immunoprecipitated and purified along with interacting proteins. The samples were then separated by SDS-PAGE. The entire lanes were excised and cut into 5 pieces and analyzed by mass spectrometry. The experiment was performed once.B) Table of selected interacting proteins detected in the control immunoprecipitation and BOD1L1 immunoprecipitations showing the number of detected peptides.C) SW620 cells were prepared as in panel (A), except that they were analyzed by Western blot. BOD1L1 was immunoprecipitated and the purified proteins were then separated by SDS-PAGE, transferred to nitrocellulose membrane and blotted as indicated. The panels were adjusted for brightness and contrast for presentation. An experiment representative of two independent biological repeats is shown.D) SW620 mitotic nuclear cell extracts were incubated with GST or GST-BOD1L fragments. Complexes were then isolated by glutathione-sepharose and analyzed by Western blot. The blot shown is representative of 2 independent experiments.

Fig 5: BOD1L1 is mutated in human cancersA) The 10,967 sequenced cancers in the TCGA were queried for the presence of mutations in the BOD1L1 gene and plotted by cancer subtype.B) Kaplan-Meier five-year overall survival by BOD1L1 alteration status with Cox-Proportional Hazards Ratios and 95% confidence intervals from models adjusted for potential confounders (age, sex [except uterine], and tumor grade or stage). A) Uterine corpus endometrial carcinoma TCGA-UCEC, n= 529; b) lung adenocarcinoma TCGA-LUAD, n = 667; c) cutaneous melanoma TCGA-SKCM, n = 448; d) stomach adenocarcinoma TCGA-STAD, n = 440; e) lung squamous cell carcinoma TCGA-LUSC, n = 487.C) U2OS, HCT116, SW620 and HeLa cells were plated. They were then transfected with control shRNA or shRNA against BOD1L1 and selected with puromycin in the presence of compounds as indicated. After 1 week of selection, the cells were fixed and stained with crystal violet. Representative images from 3 independent experiments are shown. The scale bar is 4 cm.D) Quantification of cells from (C). The staining intensity of the cells was measured using Fiji and plotted. Error bars indicate the mean +/- SEM. Statistical significance was calculated between the indicated conditions using Student’s t- test.E) Cartoon illustration showing a possible mechanism for adaptation to UMK57. UMK57 agonizes MCAK, resulting in lower k-MT stability, which is sensed by Aurora A. Aurora A then phosphorylates BOD1L1, releasing PP2A to reduce phosphorylation of the NDC80 complex, resulting in higher k-MT stability.