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. 2023 Dec 1;4(6):e439. doi: 10.1002/mco2.439

FIGURE 5.

FIGURE 5

The effect of USP22 on FKBP12 protein stabilization and ubiquitination. (A) The change in FKBP12 protein in Hep‐3B and Huh‐7 cells. (B) Silencing of FKBP12 downregulated the proteins of the mTORC1 pathway in Hep‐3B cells. (C) Elevated p‐p70S6K (T389) in oeUSP22 cells could be rescued by additional silencing of FKBP12. (D) Isolated extracts from HEK293T cells obtained for immunoprecipitation (IP) using either an anti‐Myc or anti‐Flag antibody. (E) IP assay results of endogenous USP22 and FKBP12 in SNU‐449 cells. (F) Co‐localization of FKBP12 (green) with USP22 (red) observed using confocal microscopy and indicated by yellow arrow. (G) Illustration of USP22 and its point mutants. (H) HEK293T cells transfected with FKBP12‐Flag plasmids along with NC, Myc‐USP22 (wt), and each of the point mutants. (I) Western blot and ImageJ results in Hep‐3B shUSP22 or shNC cells after cycloheximide treatment (50 μg/mL). (J) Western blot and ImageJ results in SNU‐449 oeUSP22 or oeNC cells after cycloheximide treatment (50 μg/mL). (K) Western blot showing changes in FKBP12 after MG132 (10 μM, 2 h) or 3‐methyladenine (3‐MA) (5 mM, 12 h) treatment. (L) The ubiquitination of FKBP12 using IP and western blotting in Hep‐3B shUSP22 cells. (M) The ubiquitination of FKBP12 using IP and western blotting in HEK‐293T cells with USP22 overexpression.