Extended Data Fig. 6: The TUG C-terminal product enters the nucleus and binds PPARγ and PGC-1α.
From: Insulin-stimulated endoproteolytic TUG cleavage links energy expenditure with glucose uptake
a, Nuclear fractions were prepared from quadriceps of WT, UBX, and MTKO mice that had been fasted, treated with IP injection of insulin-glucose solution or saline control, and euthanized 30 min. after injection. Immunoblots were done as indicated. b,c, Proteins were expressed by transient transfection of HEK293 cells, and immunoprecipitations (IP) and western blots (WB) were performed, as indicated. d, Recombinant proteins were produced as GST fusions, immobilized on glutathione beads, and incubated with recombinant TUG C-terminal cleavage product (residues 165-550). Bound TUG protein was eluted and western blots were performed as indicated. e, Recombinant proteins were immobilized and incubated with soluble recombinant PPARγ2 protein. Bound PPARγ2 was eluted and western blots were performed as indicated. f, Truncated forms of TUG were produced as GST fusions and the GST was cleaved off to yield soluble TUG fragments. These were incubated with immobilized GST, PGC-1α, and PPARγ2 as indicated. Bound proteins were eluted and immunoblotted as indicated. g, Peptides corresponding to the 37 residues at the N-termini of PPARγ1 or PPARγ2 were immobilized on streptavidin beads. The TUG C-terminal product (beginning with a Met residue) was expressed stably in MEFs using a retrovirus, and lysates from these cells were incubated with the beads. Bound proteins were eluted and immunoblotted. h, Peptides corresponding to the 29 N-terminal residues of PPARγ2, containing Pro12 or Ala12 residues, were immobilized on beads and incubated with lysates of MEFs expressing the TUG C-terminal product. Bound proteins were eluted and immunoblotted as indicated. i, Peptides used in (h) were incubated with HEK293 lysates, and bound endogenous (human) intact TUG was eluted and immunoblotted. j, WT and MTKO mice were treated by IP injection of insulin-glucose solution, then euthanized after 3 h. PGC-1α was immunoprecipitated from lysates of quadriceps, and immunoblots were done to detect bound PPARγ, as in Fig. 3g. The relative abundances of PPARγ in replicate experiments were quantified. N=3 biologically independent samples, presented as mean ±SEM, analyzed using a two-tailed t-test.
