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. 2023 Apr 27;14:2421. doi: 10.1038/s41467-023-38160-x

Fig. 6. POLD1 supports MYC stability by interfering with FBXW7-mediated ubiquitination.

Fig. 6

a Western blot analysis of POLD1, MYC, and FBXW7α after IgG, POLD1-IP, MYC-IP, or FBXW7α-IP in T24 and UM-UC-3 cells. MYC was used as a positive control for FBXW7α endogenous interaction. All samples were treated with MG132 (10 μM) and incubated for 6 h before harvesting cells. b Western blot of POLD1 and MYC after IgG or MYC-IP in POLD1 and/or FBXW7α knockdown T24 cells. All samples were incubated with MG132 (10 μM) for 6 h before harvest. c Western blot of ubiquitin and MYC after IgG or MYC-IP under denaturing conditions in DOX and/or siFBXW7α treatment in DOX-induced shPOLD1 T24 cells. The cells of all samples were incubated with MG132 (10 μM) for 6 h before harvest. For the precipitates, the loading volume was adjusted to be equal to the precipitated MYC (b, c). d Representative images and statistics of PLA assays of MYC and FBXW7α in 5637 cells with or without POLD1 depletion (n = 6 biologically independent experiments in each group). e Western blot of Flag-MYC, GFP-POLD1 and Myc-FBXW7α△F (F-box deletion mutant) after Flag-IP in 293T cells expressing GFP-POLD1 WT (0.1 μg, 1.0 μg, and 4.0 μg) or GFP-POLD1 L1002A (0.1 μg, 1.0 μg, and 4.0 μg) and Flag-MYC and Myc-FBXW7α△F. f The direct interaction between GST-MYC and His-POLD1 or Myc-FBXW7α△F was detected by GST pull-down assay. Recombinant GST-labeled fragments of MYC were incubated with lysates of 293T cells expressing Myc-FBXW7α△F and increased amounts of recombinant His-POLD1 (0.1, 1.0, and 3.0 μg). g The cell proliferation curve of T24 cells with POLD1 and/or FBXW7α knockdown. n = 6 biologically independent experiments in each group. h Representative images and cell numbers of Transwell assays from the indicated groups with or without POLD1 and/or FBXW7α knockdown in T24 cells (n = 3 biologically independent experiments in each group). Data are presented as the means ± SD (d, g, h). Statistical significance was determined by two-tailed Student’s T-test (d, g, h). IP-MS: Immunoprecipitation-mass spectrometry. PLA proximity ligation assay. Source data are provided as a Source Data file.