Fig 2: Expression and phosphorylation of AS160 and TBC1D1 in the infarct heart.Immunoblotting analyses of phosphorylation of AS160-Thr649 and PKB-Ser473, and total AS160, TBC1D1, PKB, GLUT4 and cTnT in the lysates (40 µg) of the infarct zone (I), border zone (B) and remote zone (R) of mouse heart 4 weeks after myocardial infarction. Flotillin-1 was used as a loading control. A, representative blots. B, quantitation of TBC1D1 signals, n = 4. Asterisk indicates p < 0.05 (infarct zone versus remote zone).

Fig 3: SBI-0206965 inhibits unstimulated and 991-stimulated glucose uptake with no apparent inhibition of TBC1D1 Ser231 phosphorylation in C2C12 myotubes.(A,B) C2C12 myotubes were pre-incubated for 30 min with vehicle (0.1% DMSO) or the indicated concentrations of SBI-0206965 followed by an additional 1 h incubation with 991 (10 µM) or vehicle. Cell lysates were subjected to immunoblot analysis using the indicated antibodies. Tubulin was used as a loading control. Representative images (A) and quantification of the indicated blots (B) from three independent experiments are shown (n = 3 per treatment). (C) C2C12 myotubes were incubated with vehicle or the indicated compounds for 1 h followed by addition of [3H]-2-deoxy-glucose and measurement of glucose uptake for 5 min (n = 8–9 per treatment). Results are expressed as mean ± SEM. *P < 0.05 (vehicle vs. indicated compound(s) treated).

Fig 4: Phosphorylations of Akt, AS160, and TBC1D1 in contralateral non-immobilized and immobilized limbs at the end of 6-h hindlimb immobilization. Muscles were dissected out at the end of 6-h unilateral hindlimb immobilization. All muscles were incubated in glucose-free medium in the absence or presence (50 µU/mL) of insulin for 20 min and then frozen. Muscle lysates were separated with SDS-PAGE and blots were analyzed for phosphorylated Akt Ser473 (A), phosphorylated Akt Thr308 (B), phosphorylated AS160 Thr647 (C), phosphorylated AS160 Ser588 (D), phosphorylated TBC1D1 Thr590 (E), and phosphorylated TBC1D1 Ser237 (F). Blots were then stripped and analyzed for total abundance of each protein. (A–C, F) Values are means ± SE (n = 7–9). (D–E) Values are means ± SE (n = 6–7). *P < 0.05 versus the contralateral non-immobilized limbs with the same insulin concentration. † P < 0.05 versus 0 µU/mL insulin.

Fig 5: Downregulation of TBC1D1 expression in Arid5b-/- GC muscles. The expression of TBC1D1 protein (a), TBC1D4 protein (b) is shown. Protein expression was normalized to the corresponding loading control (HSP90, HSP70 or vinculin), and the data are presented as the means ± SD (n = 6). ***, P < 0.001. c, d The expression of GLUT4 and GLUT1 protein in GC muscles is shown. Quantification of GLUT4 and GLUT1 protein in GC muscles is shown. (bottom) Quantitation of GLUT4 protein expression was performed, and the data are presented as the means ± SD (n = 6)