Research Paper Volume 12, Issue 23 pp 24394—24423

Figure 7. FBXW7 mediates degradation and ubiquitination of c-Myc in M2-like TAMs. (A) The protein expression of c-Myc and FBXW7 in wild-type and FBXW7-knockout primary macrophages stimulated with conditioned medium were measured by immunoblotting. (B) qRT-PCR analysis of c-Myc mRNA expression in wild-type and FBXW7-knockout primary macrophages after conditioned medium treatment. (C) Immunoblot analysis of c-Myc and FBXW7 in wild-type and FBXW7-knockout primary macrophages treated with CHX (20 μg/mL) for the indicated time period after conditioned medium pre-treatment. (D) The quantification of relative c-Myc levels in (C). (E) The protein expression of c-Myc and FBXW7 in wild-type and FBXW7-knockout primary macrophages after treatment with MG132 (10 μM) and conditioned medium. (F) The phosphodegron sequence alignment of c-Myc recognized by FBXW7 with MCL1, cyclin E, Notch1, and c-Jun. The FBXW7 phosphodegron sequence present in c-Myc is conserved across different species. Conserved residues within the degron sequences are shown in red. (G) Immunoblot analysis of BMDMs stimulated with conditioned medium for the indicated time periods and treated with MG132, followed by immunoprecipitation with specific antibody-conjugated agarose or immunoglobulin G (IgG)-conjugated agarose. (H) Immunoblot analysis of the K48 ubiquitination of c-Myc in wild-type and FBXW7-knockout BMDMs stimulated with conditioned medium. Data are shown as the mean ± SD and are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s, no significance (two-way ANOVA (B, D)).