Figure 5.

FBXW2 mediates the ubiquitination and degradation of KSRP. A) KSRP expression in FBXW2^fl/flLysm^Cre‐/− and FBXW2^fl/fl Lysm^Cre+/‐ peritoneal macrophages was tested by western blot assay (n = 5). B) Representative immunofluorescence images of KSRP and CD68 in epiWAT sections from FBXW2^fl/flLysm^Cre‐/− and FBXW2^fl/flLysm^Cre+/− mice on HFD for 12 weeks and the relative quantification (n = 10). Scale bars, 20 µm. C) Immunoblot analysis of the KSRP level in the lysates of FBXW2^fl/flLysm^Cre‐/− and FBXW2^fl/flLysm^Cre+/‐ peritoneal macrophages treated with cycloheximide (CHX; 20 µg mL⁻¹) for the indicated hours (n = 5). D) KSRP was coexpressed with empty vector or different contents of FBXW2 plasmids in Raw264.7 cells with or without MG132 treatment. The expression levels of KSRP and FBXW2 were analyzed using western blotting (n = 5). E) Real‐time qPCR assay analysis of KSRP in FBXW2^fl/flLysm^Cre‐/− and FBXW2^fl/flLysm^Cre+/‐ peritoneal macrophages (n = 5). F) HEK293T cells were transfected with Myc‐FBXW2 or Myc‐ΔF‐box‐FBXW2 plus vectors for HA‐Ub and Flag‐KSRP and then were treated with MG132. The cell lysates were subjected to immunoprecipitation with Flag M2 beads and western blotting with anti‐HA antibody (n = 5). G) Raw264.7 cells were transduced with Scr shRNA or FBXW2 shRNA plus vectors for HA‐Ub and Flag‐KSRP and then were treated with MG132. The protein extracts were subjected to immunoprecipitation with Flag M2 beads and western blotting with anti‐HA antibody (n = 5). H) Sequence alignment of KSRP with the phospho‐degron sequences recognized by FBXW2. I) HEK293T cells cotransfected with Myc‐FBXW2 plus vectors for Flag‐KSRP (WT) or Flag‐KSRP (3A). Western blot analysis for KSRP expression in the cell lysates (n = 5). J) HEK293T cells were pretransfected with Myc‐FBXW2 plus Flag‐KSRP (WT), Flag‐KSRP (3A) or HA‐Ub, and then treated with MG132. Immunoprecipitation analysis for KSRP ubiquitination with anti‐Flag‐M2 beads (n = 5). K) Myc‐FBXW2 plus Flag‐KSRP(WT) or Flag‐KSRP (3A) were first transfected into HEK29T cells. Then cells were treated with CHX (20 µg mL⁻¹) for the indicated hours. Immunoblot analysis of the KSRP levels in the cell lysates (n = 5). L) FBXW2 promotes KSRP ubiquitination via K48 linkage. Immunoblot analysis of His‐tag pull‐down and cell extracts derived from HEK293T cells transfected with the indicated constructs. The data are analyzed by Student's t test or ANOVA with the post‐hoc test and are presented as the mean ± SEM. *p < 0.05.