Fig. 3: HSP70 in interplay with STUB1 directly interacts with an N-Myc domain.

a Schematic representation of the N-Myc deletion mutants used for domain mapping. Myc boxes: I, II, III, IV; BR, basic region; HLH, helix-loop-helix; LZ, leucine zipper. b HEK293 cells were co-transfected with Flag-STUB1 and wild-type or indicated HA-N-Myc mutant constructs. The cell lysates were immunoprecipitated with the anti-Flag antibody. The red arrows mark the expected positions of the full-length or truncated N-Myc pulled down by STUB1. c HEK293 cells were co-transfected with Flag-HSP70 and wild-type or indicated HA-N-Myc mutant constructs. The cell lysates were immunoprecipitated with the anti-Flag antibody. The red arrows mark the expected positions of the full-length or truncated N-Myc pulled down by HSP70. d Alignment of the potential binding sites in N-Myc in different species to HSP70. e The Sanger Sequence chromatogram of the wild-type (WT) N-Myc plasmid and the corresponding deletion in N-Myc-ΔLILKR. f HEK293 cells co-transfected with/without His-HSP70, Flag-STUB1, and HA-WT-N-Myc or HA-N-Myc-ΔLILKR. Whole cell lysates were harvested and immunoprecipitated with the anti-HA antibody. g HEK293 cells were co-transfected with HA-WT-N-Myc or HA-N-Myc-ΔLILKR, with Flag-HSP70 or Flag-STUB1 for 3 days, and the interaction of N-Myc and HSP70 or STUB1 was determined by Proximity Ligation Assay (PLA). Scale bar represents 20 microns. h HEK293 cells were transfected with HA-WT-N-Myc or HA-N-Myc-ΔLILKR and treated with 50 μg/ml cycloheximide for 0, 30, 60, and 120 min. Whole cell lysates were separated by electrophoresis and blotted with the anti-HA antibody, and the half-life of the full-length and deleted N-Myc molecules was calculated (n = 3 independent experiments and data presented as mean ± S.D.). i HEK293 cells were transfected with HA-WT-N-Myc, HA-N-Myc-ΔLILKR, or HA-N-Myc-CLPQS, with or without Flag-STUB1 for 3 days. Total cell lysates were collected for western blotting to detect the expression of N-Myc. j HEK293 cells co-transfected with His-HSP70, HA-WT-N-Myc, HA-N-Myc-ΔLILKR, or HA-N-Myc-CLPQS and Flag-STUB1 plasmids. Whole cell lysates were immunoprecipitated with the anti-N-Myc antibody. Source data are provided as a Source Data file.