Figure 3.
TGFB3-AS1 interacts with Rap1a and protects it from ubiquitin-proteasome-mediated degradation in macrophages
(A) Schematic of RNA pull-down coupled with mass spectrometry analysis for identifying proteins interacting with TGFB3-AS1 in macrophages. (B) Separation of proteins pulled down by TGFB3-AS1 or its antisense RNA control (antisense TGFB3-AS1) using silver-stained SDS-PAGE. The black frame indicates the specific bands of proteins pulled down by TGFB3-AS1 compared with the antisense TGFB3-AS1 control. (C) The top 18 significantly enriched proteins in the RNA pull-down mass spectrometry analysis are listed in the table, with their score, matches, and emPAI displayed. (D) Whole-cell lysate of macrophages was immunoprecipitated with biotinylated TGFB3-AS1 or its antisense control (antisense TGFB3-AS1) followed by immunoblotting with the antibodies against Rap1a, Vim, and Hist1h4. (E) qRT-PCR examined enrichment of TGFB3-AS1 in Rap1a-immunoprecipitated RNA complexes using Rap1a antibodies, isotype identical IgG served as a negative control. (F and G) The mRNA and protein expression of Rap1a in macrophages transduced with either lenti-TGFB3-AS1 or TGFB3-AS1 siRNAs (TGFB3-AS1-siRNA1 and TGFB3-AS1-siRNA2) were detected by qRT-PCR and western blot, respectively. (H) Left panel: western blot was used to detect the expression of Rap1a in macrophages treated with lenti-TGFB3-AS1 and 20 μg/mL cycloheximide (CHX) for various times (0, 15, 30, 60, 120, and 240 min). Right panel: the quantification of Rap1a protein levels in the upper panel. T1/2 denotes an estimated half-life. (I) Western blot was used to detect the expression of Rap1a in macrophages treated with TGFB3-AS1-siRNA or MG132 (5 μmol/L, 24 h). (J) The ubiquitination level of Rap1a was measured by immunoprecipitation with anti-Rap1a antibody and immunoblotting with anti-Ub antibody in macrophages transduced with TGFB3-AS1-siRNA or TGFB3-AS1-sc in the presence of MG132 with or without Hcy. ∗p < 0.05, compared with TGFB3-AS1-sc, lenti-GFP, or 100 μmol/L Hcy. ∗∗p < 0.01, compared with IgG. #p < 0.05, ##p < 0.01, compared with the group of lenti-GFP + 100 μmol/L Hcy or TGFB3-AS1-sc + 100 μmol/L Hcy.