FIGURE 4.

MAFG‐AS1 can stabilize Hu antigen R (HuR) by recruiting the deubiquitinating enzyme USP5. A, Western blot analysis of HuR expression in bladder urothelial carcinoma (BUC) cells. B, Western blot analysis of HuR expression in cancer and normal tissues of bladder. C, Immunohistochemical detection of HuR expression in cancer and normal tissues of bladder. D, Western blotting was used to detect the expression level of HuR in each experimental group. E and F, T24 and RT4 cell lines were cultured with cycloheximide (10 μg/mL), adding MG132 (20 μM) or transfected with sh‐MAFG‐AS1, and cell lysates were analyzed by Western blotting. G, Detection of HuR protein expression by Western blotting after knockdown of deubiquitinating enzyme in T24 and RT4 cell lines. H, Western blotting shows the expression level of HuR in T24 and RT4 cells after transfected with MAFG‐AS1 or MAFG‐AS1 and si‐USP5. I, The HDOCK website predicts that HuR can bind to USP5. J, USP5 expression was determined by Co‐IP using HuR‐antibody to precipitate binding proteins. K, The level of ubiquitination of HuR was detected by Co‐IP after overexpression of MAFG‐AS1 or overexpression of MAFG‐AS1 and knocking down USP5 in T24/RT4 cells. Bars represent standard deviation, ns P > .05, *P < .05, **P < .01, ***P < .001