Skip to main content
. Author manuscript; available in PMC: 2015 Jun 3.
Published in final edited form as: Cell Metab. 2014 Apr 17;19(6):967–980. doi: 10.1016/j.cmet.2014.03.018

Fig. 6. Phosphorylation of Grb10 plays a critical role in its feedback inhibition of mTOR signaling in brown adipocytes.

Fig. 6

(A) The immunoprecipitation (IP) of HA-Grb10 and Co-IP of raptor in starved brown adipocytes transiently expressing HA-tagged Grb10 treated with or without 10 nM insulin at different times as indicated. (B) The IP of HA-Grb10 and Co-IP of raptor and IRβ in starved brown adipocytes transiently expressing HA-tagged Grb10 treated with or without 5 nM rapamycin for 60 min, followed with or without 10 nM insulin for 10 min. (C) Insulin-stimulated phosphorylation of Akt and S6K and the expression of Grb10 in HEK293 cells transiently expressing wild-type Grb10 (WT), Grb10S501/503A (AA), or Grb10S501/503D (DD) treated with or without 10 nM insulin for 20 min. (D) The IP of HA-Grb10 and Co-IP of raptor and IRβ in starved HEK293 cells transiently expressing wild-type HA-tagged Grb10 (WT), Grb10S501/503A (AA), or Grb10S501/503D (DD) treated with or without 10 nM insulin. (E) The immunoprecipitation (IR)of mTOR and Co-IP of raptor in starved Grb10-suppressed brown preadipocytes (RNAi) and scramble control cells.(F) The IP of raptor and Co-IP of mTOR in starved HEK293 cells transiently expressing wild-type (WT) Grb10, Grb10S501/503A (AA), or Grb10S501/503D (DD) mutant treated with or without 10 nM insulin for 20 min. (G) The effect of overexpression of wild-type (WT), Grb10S501/503A (AA), or Grb10S501/503D (DD) on CL316343 (CL)-induced thermogenic and lipolytic gene expression in primary brown adipocytes isolated from Grb10fKO mice. Cells were treated with or without 1μM CL316243 for 22 hours. (H) The effect of overexpression of WT, AA, or DD mutants of Grb10 on isoproterenol (Iso)-induced glycerol released in primary brown adipocytes isolated from Grb10fKO mice. Data are presented as mean ± S.E.M. *<0.05. (I) Fatty acid released from Grb10-deficient primary brown adipocytes transfected with vector or vectors encoding WT or the phosphorylation site mutants of Grb10 (AA or DD). Data are presented as mean ± S.E.M. *<0.05.