Fig. 6: Myristoylated Eepd1 localizes to the cell membrane and forms an association with PKA.
From: Myristoylated Eepd1 Enhances Lipolysis and Thermogenesis through PKA Activation to Combat Obesity
a Flowchart for measuring Eepd1 myristoylation levels in vivo using MS43. b MS results of Eepd1 myristoylation in iWAT of C57BL/6 J mice under 8 °C and 25 °C conditions (n = 4). c Flowchart for click chemistry-based myristoylation measurement44. d Click-chemistry-based analysis of Eepd1 myristoylation in C3H10T1/2-derived adipocytes infected with adenovirus expressing Eepd1(WT) or Eepd1(G2A) for 48 h. TCL, total cell lysate. e Click-chemistry-based analysis of the Eepd1 myristoylation in C3H10T1/2-derived adipocytes infected with adenovirus expressing Eepd1(WT) or Eepd1(G2A) for 48 h and then incubated with ISO, NE or CL316243 (CL) for 6 h. f Immunoblots of indicated proteins in mature C3H10T1/2-derived adipocytes infected with indicated adenovirus for 24 h and then incubated with NE for the indicated time. g Click-chemistry-based analysis of Eepd1 myristoylation in iWAT of WT and ob/ob mice. h Immunoblots of Eepd1 in EKO primary adipocytes infected with Adv-Eepd1(WT)-EGFP or Adv-Eepd1(G2A)-EGFP for 48 h, then treated with cycloheximide (CHX) for the indicated time. i Densitometry quantification of Eepd1/β-actin. The dotted line shows Eepd1 half-life (n = 3 technical replicates/group). j Immunoblots of Eepd1 in plasma membrane and cytoplasm of EKO primary adipocytes infected with Adv-Eepd1(WT)-EGFP or Adv-Eepd1(G2A)-EGFP for 48 h. E-cadherin and GAPDH were used as markers for cell membrane and cytoplasm. k, l Representative images of immunofluorescence for Eepd1 (green) in C3H10T1/2-derived adipocytes (k) and HeLa cells (l) infected with either Adv-Eepd1(WT)-EGFP or Adv-Eepd1(G2A)-EGFP. Scale bar, 20 μm. m–q Co-IP assay showing Eepd1 interaction with PKA subunits in 293 T cells. STE, short-time exposure. LTE, long-time exposure. r Co-IP assay showing Eepd1 interaction with HSL, p38 MAPK and CREB in adipocytes overexpressing Eepd1-Flag. s Representative immunofluorescence images of Eepd1 and PKA-RIIα in C3H10T1/2-derived adipocytes infected with the indicated adenovirus. Scale bar, 20 μm. Data presented as mean ± SEM. Significance was assessed by unpaired two-tailed Student’s t test (b) or two-way ANOVA (i). Source data are provided as a Source Data file.
