Fig 2: Hyperphosphorylated MYC retains interaction with MAX but is compromised for chromatin binding. a MCF10A cells were treated with Calyculin A (50 nM) or vehicle control for 30 min, lysed, and immunoprecipitated with MAX rabbit or IgG rabbit antibody. The input and IP was resolved by SDS-PAGE and immunoblotted with MYC mouse antibody. Shown is a representative image of n = 3. b For ChIP analysis, MCF10A cells were treated with MG132 (10 µM) for 4 h ± Calyculin A (50 nM) for 30 min, and lysate incubated with MYC rabbit antibody along with IgG antibody as a control. ChIP’d DNA was quantified by qPCR for MYC target genes promoters of CAD, CDK4, LDHA, and NCL, and chr6 (negative control). c For nuclear and cytoplasmic fractionation, MCF10A cells were treated with MG132 (10 µM) for 4 h ± Calyculin A (50 nM) for 30 min (left panel), or transfected with 10 nM siRNA directed against PP1a, ß, and ? or universal scramble negative control by calcium phosphate for 44 h and then treated with MG132 (10 µM) for 4 h (right panel). Lysates were used for fractionation, and immunoblotted with indicated antibodies. d MCF10A cells carrying inducible FLAG-tagged EV, MYC wt, MYC12A, or MYC12D were treated with 4-OHT (24 h) to induce expression, treated with MG132 (10 µM) for 4 h, and then lysed for immunoblot analysis (top) or for ChIP-qPCR with FLAG or IgG antibody (bottom) as outlined in b above. For b, d: shown are % input mean (MYC and FLAG ChIP: n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Bonferroni test. Not statistically significant = ns. Error bars represent s.d. e Proposed model of PP1/PNUTS regulation of MYC hyperphosphorylation in the control of chromatin binding and degradation

Fig 3: MYC is post-translationally modified and interacts with the PP1/PNUTS phosphatase complex. a Cell lysate from growing MCF10A cells was immunoprecipitated with MYC monoclonal mouse antibody, resolved by 2D gel electrophoresis (7 cm, pH 4–7 IPG strip; 10% SDS-PAGE), transferred onto nitrocellulose membrane and immunoblotted with MYC polyclonal rabbit antibody. Representative image of n = 3. Shown is lower exposure (top panel) and higher exposure (bottom panel) x-ray film images. Arrows show numerous high molecular weight MYC spots. b Shown are the gene names and spectral counts of selected known and novel MYC interacting proteins identified by conducting MYC-BioID MS in HeLa cells. A and B are biological replicates with two technical replicates shown for each. Total spectral counts and SAINT analysis score are presented. Significance is achieved with SAINT > 0.8. c Schematics of protein phosphatase 1 (PP1) and protein phosphatase 1 nuclear-targeting subunit (PNUTS). The catalytic subunit of PP1 a, ß, or ? interacts with a regulatory subunit such as PNUTS, which forms a heterodimeric holoenzyme that interacts with PP1:PNUTS substrates