Fig. 4: VRK2 reduces FBXO24-mediated polyubiquitination degradation of the MYC K323 by activating phosphorylation of MYC at 281 and 293. | Nature Communications

Fig. 4: VRK2 reduces FBXO24-mediated polyubiquitination degradation of the MYC K323 by activating phosphorylation of MYC at 281 and 293.

From: VRK2 targeting potentiates anti-PD-1 immunotherapy in hepatocellular carcinoma through MYC destabilization

Fig. 4: VRK2 reduces FBXO24-mediated polyubiquitination degradation of the MYC K323 by activating phosphorylation of MYC at 281 and 293.The alternative text for this image may have been generated using AI.

a, b CHX chase assays in Hep3B (VRK2 overexpression) and MHCC97H (VRK2 knockout) cells treated with CHX (20 μM). Quantified MYC levels normalized to GAPDH (bottom). c Co-IP of VRK2, MYC, and Ub in HLF cells overexpressing VRK2 and transfected with MYC-tagged Ub plasmids, with MG132 (10 μM, 4 h). d Co-IP of Flag-VRK2, HA-MYC, and MYC-tagged Ub K48 in HEK293T cells (MG132-treated). e Western blot analysis of HA-MYC in HEK293T were transfected with Flag-FBXO24 WT, Flag-FBXO24ΔF-box and HA-MYC plasmids. f, g Immunoprecipitation assay of interaction among MYC, VRK2, or FBXO24 in HLF (up) and MHCC97H cells (bottom). h Co-localization analysis of MYC and FBXO24 in MHCC97H cells. Scale bar: 20 μm. The nucleus was stained with DAPI (n = 3 independent experiments). i Western blot analysis of VRK2, MYC, and FBXO24 in knockout VRK2 MHCC97H cells with interference with FBXO24 expression using siRNA. j Co-IP of MYC, VRK2, or FBXO24 in HEK293T with CIAP treatment. k Co-IP of Flag-VRK2, HA-FBXO24, and MYC-tagged MYC in HEK293T (MG132-treated). l Co-IP of VRK2, FBXO24, and MYC in HLF cells overexpressing oe-VRK2 WT/MUT (MG132-treated). m Co-IP of VRK2, FBXO24, and MYC in MHCC97H cells with sgCON/sgVRK2 (MG132-treated). n–p Co-IP of Flag-FBXO24, MYC-tag Ub, HA-MYC WT or HA-MYC S281A/S281D (n) or HA-MYC S293A/S293D (o) or HA-MYC S281A + S293A(2SA)/S281D + S293D(2 SD) (p) in HEK293T cells and incubated with MG132 (10 μM) for 4 h. For Western blot experiments, GAPDH was used as a loading control (a–e, i–p). The cells involved in the above experiments were harvested for western blotting with the indicated antibodies (n = 3 independent experiments). Source data are provided as a Source Data file.

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