Fig. 4: HDSP interacts with MECOM and prevents it from ubiquitination-mediated degradation.

a In SGC-7901 cells with stable overexpression of HDSP-Flag, the IP enrichment of the target protein interacting with HDSP was assessed by silver staining. b Bidirectional Co-IP was employed to investigate the interaction between HDSP and MECOM in GC cells. c The interaction between HDSP and MECOM in GC cells was detected by PLA. The experiment was repeated 3 times independently with similar results. Scale bar: 10 μm. d The swMATH software provides the most likely docking method for HDSP and MECOM. e Schematic diagram of HDSP and MECOM truncation. HDSP is evenly divided into N-terminal and C-terminal segments, while MECOM is divided into four segments corresponding to its four domains. f The binding sites between HDSP and MECOM were verified through Co-IP experiments using their different truncated forms. g After the expression and protein purification of MECOM-His and HDSP-GST, the interaction between HDSP and MECOM in vitro was detected using a GST pull-down assay. h Western blot analysis was conducted to assess MECOM expression levels in GC cells with stable low or overexpression of HDSP. i GC cells were treated with cycloheximide (CHX, 10 µM), and MECOM protein levels were assessed by western blot at 0, 6, 12, and 18 h. j GC cells were treated with either MG132 (10 mM) or CQ (20 µM) for 6 h, following which the expression levels of MECOM were evaluated using western blot. k After co-transfection of HA-Ub and MECOM-Flag, HEK-293T cells were treated with MG132, and the ubiquitination level of MECOM was assessed using the Co-IP. l Co-IP was employed to identify the type of ubiquitination modification of MECOM mediated by HDSP. m, n Co-IP was utilized to assess the ubiquitination modification level of MECOM following knockdown or overexpression of HDSP. All western blot assay was repeated 3 times independently with similar results. Source data are provided as a Source data file.