Fig. 5: Myristoylation of Gly2 is required for Eepd1-mediated thermogenesis and lipolysis.

a Sequence alignment comparison of Eepd1’s first 10 residues across species. b-d Immunoblots of indicated proteins in C3H10T1/2-derived adipocytes reverse-transfected with mock or Eepd1 plasmids for 24 h and then treated with inhibitors for another 24 h. e Immunoblots of phospho-PKA substrates (RRXS*/T*) and HSL in C3H10T1/2-derived adipocytes reverse transfected with indicated plasmids for 48 h. f OCR in C3H10T1/2-derived adipocytes transfected with mock, Eepd1 or Eepd1 mutant plasmids for 48 h (n = 7 biological replicates). g, h Glycerol and NEFA release in C3H10T1/2-derived adipocytes transfected with plasmids for 48 h (n = 4 biological replicates). i NMT1 and NMT2 mRNA levels in human adipose tissues. BMI < 30, n = 14; BMI > 30, n = 28. j–l Immunoblots of Nmt1 and Nmt2 in eWAT from DIO, db/db and ob/ob mice comparing to respective littermate controls. m, n Nmt1 and Nmt2 mRNA levels in iWAT and eWAT from 8-week-old male C57BL/6 J mice exposure to 4 °C (n = 5) or room temperature (RT) (n = 4) for 3 days. o Nmt1 mRNA level in C3H10T1/2-derived adipocytes incubated with ISO for 6 h (n = 12 biological replicates). p Immunoblots of indicated proteins in primary adipocytes transfected with siRNAs for 24 h and then infected with Adv-Ctl or Adv-Eepd1 for another 48 h. q Quantification of p-HSL/HSL (n = 4 technical replicates/group). r Immunoblots of indicated proteins in C3H10T1/2-derived adipocytes transfected with siRNAs for 24 h and infected with adenovirus for another 24 h. s OCR in C3H10T1/2-derived adipocytes transfected with siRNAs for 24 h and then infected with adenovirus for another 24 h (n = 7). Data presented as mean ± SEM. Significance was assessed by unpaired two-tailed Student’s t test (i, n, o), one-way ANOVA (f, g, h, q, s) or two-sided Mann-Whitney U test (m). β-actin and GAPDH were used as loading controls for immunoblots. 2-HM, 2-hydroxy Myristic Acid methyl ester. 2-BP, palmitoylation inhibitor 2-bromopalmitate. Source data are provided as a Source Data file.