Fig 2: HIV-1 targets UBA2, a subunit of the SUMO E1–activating enzyme.(A) HEK293 cells or CD4+ Jurkat T lymphocytes were transfected or electroporated with the lentiviral vector pfNL43-dE-EGFP encoding HIV-1 as described above, followed by lysis at indicated times post-transfection to assess the level of free (unconjugated) SUMO1 and SUMO2/3 proteins by Western blot. (B) Western blots show UBA2, SAE1 and UBC9 protein levels in HEK293 and Jurkat cells. Cells were lysed and proteins were analyzed at indicated times after transfection or electroporation with HIV-1. HIV-1 specifically down-regulates the host UBA2 protein, a subunit of the heterodimeric SUMO E1 enzyme. Densitometric quantifications of UBA2, SAE1, and UBC9 signals were normalized to tubulin expression, which serves as a loading control. Data are presented as mean ± SEM (n = 3 for HEK293 and Jurkat cells), asterisks denote statistical significance (P-values were calculated using t test assuming unequal variances, ns, not significant). Control, cells transfected with an empty vector backbone devoid of HIV-1 genes; HIV, cells expressing the HIV-1 genome.

Fig 3: The SUMOylation reduced the activity of PPAR?2 through K107 site and weakened adipogenic differentiation. (A) The SUMOylation of PPAR?2 was relied on Lys residues of 107 and 395. Flag-PPAR?2, Flag-PPAR?2-K107R, ubc9 and RH-SUMO3 were co-expressed in HEK293T cells and co-IP assays were performed with Flag and RGS antibody. (B) The expression of PPAR?2 in ST2 cells transfected by Flag-PPAR?2 for 24h (a) and NC-siRNA and PPAR?2-siRNA for 36 h (b) were detected by Western Blot. (C) NC-SiRNA, PPAR?2–SiRNA, Flag-PPAR?2, and Flag-PPAR?2-K107R were transfected in mouse mesenchymal stromal cell line ST2 cells and the expression of adipogenic marker genes C/EBPa (a), adiponectin (b), and FABP4 (c) was analyzed by Q-PCR after 7-day adipogenic induction. (D) Flag-PPAR?2, Flag-PPAR?2-K107R, and RH-SUMO3 were transfected in ST2 cells and the expression of adipogenic marker genes C/EBPa (a), adiponectin (b), and FABP4 (c) was analyzed by Q-PCR after 7-day adipogenic induction. Data are shown as the mean ± SEM. **p < 0.01, ***p < 0.001; ****p < 0.0001. All the data were obtained from at least three independent experiments.

Fig 4: a-tubulin is SUMOylated in cells and in vitro. (A) Immunoprecipitates with a-tubulin Ab from HEK293 cells expressing Flag-SUMO1, Flag-SUMO2, or Flag-SUMO3 were subject to immunoblotting (IB) and probed with Flag and a-tubulin (a-Tub) Abs. (B) Endogenous a-tubulin in HEK293 cells was immunoprecipitated and probed with SUMO1 Ab. (C) Immunoprecipitates with a-tubulin Ab from HEK293 cells expressing HA-Ubc9 were probed with HA and a-tubulin Abs. (D) Coomassie blue staining of purified mouse brain tubulin including a-tubulin and ß-tubulin, indicated by arrows. (E) Coomassie blue staining of purified GST-SAE2/1, GST-Ubc9, His-SUMO1GG, and His-SUMO1?GG. Asterisk indicates the band of purified protein. (F) In vitro SUMOylation assay using purified GST-SAE2/1, GST-Ubc9, His-SUMO1GG, and brain tubulins. (G) Ratio of density of SUMOylated bands to unSUMOylated bands. (H) Purified tubulin was in vitro SUMOylated and probed with a-tubulin Ab. (I) In vitro SUMOylation assay using soluble tubulins and MTs. (J) Immunoprecipitates with ß-tubulin Ab from HEK293 cells expressing Flag-SUMO1, Flag-SUMO2, or Flag-SUMO3 were probed with Flag and ß-tubulin Abs. (K) Purified tubulin was in vitro SUMOylated and probed with ß-tubulin Ab. (L) Endogenous ß-tubulin in HEK293 cells was immunoprecipitated and probed with SUMO1 Ab. The experiments were repeated three times.

Fig 5: Conditional SUMO knockdown attenuates SUMO pathway activity.(A) U2OS cells were infected with Dox inducible SAE2 shRNAs (sh1-sh4) or control shRNA (c). Cells were treated with Dox for 5 days (+) or untreated (-). Protein lystes were immunoblotted for indicated proteins. TUBULIN serves as loading control. (B-D) U2OS cells expressing conditional SAE2 shRNAs were treated with Dox and immunoblotted with RanGAP1 (B), SUMO1 (C) and SUMO2/3 (D) antibodies.