Fig. 5: POLD1 is a direct binding partner of MYC.

a Western blot analysis of GFP-POLD1 and HA-MYC after GFP-IP or HA-IP in 293 T cells. The input was 10% of the extract used for the IP. b Western blot analysis of POLD1, MYC, and MAX after IgG, POLD1-IP, MYC-IP, or MAX-IP in T24 (top panel) and 5637 (bottom panel) cells. MAX was used as a positive control for MYC endogenous interaction. c Schematic diagram of various MYC truncations in the Co-IP assays (top panel). Western blot analysis of GFP-POLD1 and HA-MYC after HA-IP in 293T cells (bottom panel). d Schematic diagram of various MYC deletion mutations in POLD1 binding assays (top panel). Western blot analysis of GFP-POLD1 and Flag-MYC after Flag-IP in 293T cells (bottom panel). e MST analysis was used to measure the binding affinity of lysates of overexpressed GFP-POLD1 in 293T cells and the MYC-MB1 (WT) peptide (Graph shows mean ± SD from three biologically independent experiments in each group). Here, GFP-FBXW7α was used as a positive control. The centerline indicates the median, bounds of box = 25th and 75th percentiles, bars = 10th and 90th percentiles, whiskers = min to max. f Schematic diagram of various recombinant full-length and fragment GST-MYC proteins in the GST pull-down assays (left panel). g Western blot analysis of His-POLD1, GST-MYC after GST pull-down assays (right panel). The red arrows indicate the theoretical location of the full-length and fragment GST-MYC or GST. h Confocal microscopy images of PLA of the POLD1 and MYC interaction in 5637 cells. Data are representative images from three independent assays. MBI MYC homology box 1, MBII MYC homology box 2, MBIII MYC homology box 3, MBIV MYC homology box 4, BR-HLH-LZ basic region (BR), and helix-loop-helix-leucine zipper (HLH-LZ) domain, ΔMB MYC box deletion mutants; * represents the heavy chain and ** represents the light chain. Source data are provided as a Source Data file.