Figure 5.

Ubiquitination of FSP1 in vitro. (A) Positions of identified ubiquitination sites in TCMK-1 cells determined using mass spectrometry. Sequences marked with light red indicate ubiquitination positions. (B) TCMK-1 cells were treated with cycloheximide (CHX) at different time points. Cell lysates were harvested over 10 h and analyzed on SDS/PAGE followed by western blotting. (C) FSP1 protein from (B) half-life. (D) Plasmids encoding FSP1 and HA-ubiquitin (Ub) were co-transfected into TCMK-1 cells. Then, cell lysates were immunoprecipitated with anti-FSP1 or anti-HA and Western blot analysis was performed for FSP1 and HA showing ubiquitinated species of FSP1. (E) FSP1 protein level with different concentrations of MG132 was analyzed. (F) TCMK-1 cells were treated with MG132 at different concentrations. FSP1 mRNA levels were analyzed using real-time PCR. Results are shown as mean ± standard deviation from three independent experiments. Significance levels were estimated using ANOVA. ∗∗P < 0.01. P < 0.05 was considered statistically significant. ns, no significant difference. (G) Total lysates were analyzed for FSP1 and ubiquitinated proteins by immunoblotting using anti-FSP1 and anti-Ub antibodies. The decrease in FSP1 protein observed upon CD36 overexpression was abolished by MG132, while the ubiquitination of FSP1 was increased. (H) IP with FSP1 antibody and western blotting with anti-Ub show that ubiquitination of FSP1 was higher in CD36-overexpressing plus MG132-treated cells vs. control untreated cells as well as empty vector-transfected plus MG132-treated cells. IgG was used as a negative control.