Fig. 5: ISM1 does not activate AKT phosphorylation in 3T3-F442A cells.
From: Crystal structure of Isthmin-1 and reassessment of its functional role in pre-adipocyte signaling
a IMAC purified TL-AMOP expressed from Expi293 cells shows stimulation of AKT phosphorylation at S473 in a dose-dependent manner in the preadipocyte 3T3-F442A cell line. Phosphorylation of endogenous AKT at S473 (pAKT473) and total AKT were assessed by immunoblotting and quantified. A representative blot is shown and quantification of three independent biological repeats are presented as mean ± s.d. b Activity of IMAC plus SEC purified TL-AMOP is diminished. A representative blot is shown and quantification of three independent biological repeats are presented as mean ± s.d. c IMAC pulldown of media from Expi293 cells either expressing ISM1-FL protein or not expressing recombinant protein (Expi293med) as a negative control. Both ISM1-FL and the negative control exhibited activity on 3T3-F442A AKT phosphorylation in a dose-dependent manner. A representative blot is shown and quantification of two independent biological repeats are presented as mean ± s.d. d SEC profiles of ISM1-FL (black line) and Expi293med (gray dash line) are shown. Fractions were assessed by SDS-PAGE and combined as frac1 (10.4–11.4 mL) and frac2 (15.9–16.9 mL). e The activity of the fractions of ISM1-FL and Expi293med on 3T3-F442A AKT phosphorylation indicates pure ISM1-FL protein (ISM1-FL F1) has limited activity but F2 of both ISM1-FL and Expi293med have potent activity. A representative blot is shown and quantification of four independent biological repeats are presented as mean ± s.d. f The activity of twin-strep tagged ISM1-FL purified by Strep-Tactin resin (ISM1-FLts) compared to ISM1-FL purified by IMAC on 3T3-F442A AKT phosphorylation. A representative blot is shown and quantification of three independent biological repeats are presented as mean ± s.d. Source data are provided as a Source Data file.
