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. 2023 Aug 6;10(28):2302130. doi: 10.1002/advs.202302130

Figure 7.

Figure 7

Palmitoylation‐stabilized IRHOM2 suppresses its ubiquitination and TRIM31‐triggered degradation. a) The THLE2 cells were transfected with indicated vectors for following coimmunoprecipitation (Co‐IP) assay. Co‐IP detection of the interaction of IRHOM2 with ZDHHC3 in THLE2 cells transfected with Flag‐IRHOM2 and HA‐ZDHHC3 or HA‐IRHOM2 and Flag‐ZDHHC3 plasmids. Immunoblotting probed with anti‐HA or anti‐Flag antibody (upper). Representative immunoblotting bands for GST precipitation showing ZDHHC3‐IRHOM2 binding by treating purified His‐ZDHHC3 with purified GST‐IRHOM2 or by treating His‐IRHOM2 with purified ZDHHC3‐GST in vitro (lower). Purified GST was regarded as a control. b,c) Schematic of human full‐length and truncated ZDHHC3 and IRHOM2 (b), and representative western blotting mapping assay indicating the interaction domains of ZDHHC3 and IRHOM2 (c). d) IP detection in THLE2 cells showing the effects of palmitoylation inhibitor 2‐BP challenge and ZDHHC3 deficiency on the binding between IRHOM2 and other interested proteins with known function, and representative immunofluorescence images of TRIM31 and MAP3K7 coexpression (upper), and K48‐linked ubiquitination of IRHOM2 (lower) (n = 5 images per group). Scale bars, 10 µm. e) ZDHHC3 structure showing the catalytic activity motif mutant (C157A) of DHHC domain in different species. f) IP detection in THLE2 cells showing the effects of ZDHHC3 knockout and ZDHHC3 with C157A mutant on the binding between IRHOM2 and IRHOM2 downstream signaling factors, i.e., MAP3K7 and TRIM31, and representative immunofluorescence images of TRIM31 and MAP3K7 coexpression (left), and K48‐linked ubiquitination of IRHOM2 (right) (n = 5 images per group). Scale bars, 10 µm. g) Immunoblotting assay showing the involvement of TRIM31 in depalmitoylation‐triggered proteasome degradation of IRHOM2 in ZDHHC3‐deleted THLE2 cells (upper) or ZDHHC3 (C157A)‐transfected THLE2 cells (middle) (n = 4 samples per group). THLE2 cells expressing IRHOM2 or IRHOM2‐Ub were transfected with AdshTRIM31, followed by a specific antibody for IRHOM2 used to detect the IRHOM2 or IRHOM2‐Ub expression. Negative control (NC) or empty vector (EV) were used as control. This experiment was repeated three times.