Fig. 4
SUMOylation stabilizes AnxA6 protein to counteract ubiquitin-mediated degradation. (A) The SUMO1-modified AnxA6 was detected in HEK293T cells under co-transfection of plasmids pHA-Ubc9 and pMyc-SUMO1. The SUMOylated AnxA6 was captured by IP with anti-AnxA6 in HEK293T cells. (B-C) The endogenous SUMOylated AnxA6 was measured in HEK293T cells. The SUMOylated AnxA6 was captured by IP with anti-AnxA6 (B) or anti-SUMO1 (C) antibody, then respectively immunoblotted with anti-SUMO1 or anti-AnxA6 antibody. The normal IgG was a nonspecific binding control for IP. S-AnxA6: Flag-tagging SUMOylated AnxA6. (D) Antagonism between SUMOylation and ubiquitination of AnxA6 protein. After the target plasmids were transfected into HepG2 cells for 48 h, 20 μM MG132 as a proteasome inhibitor was added to incubate with cells for 6 h, and then cellular lysates were used to capture AnxA6 by IP, from which the enriched AnxA6 was detected through western blot. (E) AnxA6 was decreased under ML-792 inhibitor exposure in HepG2 cells. HepG2 cells were treated with 10 μM ML-792 inhibitor for 48 h, and followed to perform immunofluorescence assay. The protein expression of AnxA6 was analyzed using the Pixel Intensity Spatial Correlation Analysis of software Image J. E-cadherin was taken as a positive control for cell membrane expression. Scale bars, 20 μm. Nuclei were counterstained with DAPI (blue). (F) The level of AnxA6 ubiquitination was detected in TPA-induced HEK293T cells under co-transfection of plasmids pFlag-AnxA6, pHA-ubiquitin and pMyc-SUMO1. (G) AnxA6 SUMOylation was reduced in TPA-induced EMT cells. The endogenous AnxA6 protein in TPA-induced cells was enriched from cell lysate by IP, in which the SUMOylated AnxA6 level was analyzed through western blot using SUMO1 antibody (up). The quantitative analysis of the SUMOylated AnxA6 levels in TPA induced-LO2 and HepG2 cells were also shown. The bar chart represented the ratio of SUMOylated AnxA6 to the total AnxA6 protein in the IP elution solution (bottom). *P < 0.05. (H) Cellular endogenous SUMO1 was enriched by IP to detect SUMOylated AnxA6 in TPA-induced HepG2 cells. (I) The native SUMO1-modified AnxA6 level was detected in MHCC-97 L and MHCC-97 H HCC cells with low/high metastasis abilities. Cellular endogenous AnxA6 was enriched by IP to detect SUMOylated AnxA6 against anti-SUMO1 antibody (up). The bar chart represents the ratio of SUMOylated AnxA6 versus the total AnxA6 protein in the IP elution solution (bottom). **P < 0.01