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. 2019 Sep 16;129(10):4477–4491. doi: 10.1172/JCI127374

Figure 4. Neddylation of SRSF3 in response to palmitic acid.

Figure 4

HepG2 cells (A) and primary hepatocytes (B) were treated with 10 μM MG132 (MG) for 2 hours. Cell lysates were immunoprecipitated (IP) with anti-NEDD8 antibodies and then immunoblotted (IB) for SRSF3. Input indicates input control, Co-IP indicates the coimmunoprecipitated proteins. The unmodified SRSF3 and NEDD8-SRSF3 conjugate are indicated by arrows. (C) HepG2 cells were transfected with Flag-tagged SRSF3 and then treated with 10 μM MG132; cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted for NEDD8. Monomeric NEDD8 and the NEDD8–Flag-SRSF3 conjugate are indicated by arrows. (D) HepG2 cells were transfected with Flag-tagged SRSF3 and then treated with 10 μM MG132 and 500 μM palmitic acid (PA); cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted for NEDD8. (E) HepG2 cells were transfected with Flag-tagged SRSF3 and then treated with the NEDD8-activating enzyme 1 inhibitor 3 mM MLN4924 (MLN49) for 1 hour and then PA for 12 hours, and cell lysates immunoprecipitated with an anti-Flag antibody and then immunoblotted for NEDD8. The NEDD8–Flag-SRSF3 conjugate is indicated by an arrow in D and E. (F) HepG2 cells were cotreated with the antioxidant N-acetyl-cysteine (NAC) and PA for 12 hours; cell lysates were immunoprecipitated with anti-NEDD8 antibody and then immunoblotted for SRSF3. NEDD8-SRSF3 is indicated by an arrow. (G) Primary hepatocytes or HepG2 cells were treated with 3 mM MLN49 for 1 hour and then PA for 12 hours, and cell lysates were immunoblotted for SRSF3. (H) Primary hepatocytes or HepG2 cells were treated with 3 mM MLN49 for 1 hour and then PA for 12 hours; splicing of the Fn1-EDA exon 33, Myo1b–exon 23, and Slk–exon 13 was analyzed by RT-PCR. (I) HepG2 cells were treated with 3 mM MLN49 and 1 mM MLN7243 (MLN72, ubiquitin-activating enzyme 1 inhibitor) for 1 hour and then PA for 12 hours; cell lysates were immunoblotted for SRSF3. Results are presented as mean ± SEM (n = 3). *P < 0.05 by 1-way ANOVA.