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. 2015 Aug 20;470(Pt 2):207–221. doi: 10.1042/BJ20150404

Figure 1. Effect of GSK3 inhibitors on mTORC1 signalling in L6 myotubes, HeLa and HEK293T cells.

Figure 1

(A–C) Cells were held in EBSS containing or lacking AAs for 1 h or alternatively having been depleted of AAs incubated in EBSS containing a 1× AA mix (refeed) for 15 min. Cells were incubated with 100 nM rapamycin, 50 μM SB415286 or 30 μM roscovitine for 15 min prior to and for 15 min during the AA-refeed period. Cells were lysed and 30 μg of lysate protein was analysed by immunoblotting. Blots shown in (A–C) are representative of a minimum of three independent experiments and histograms (means ± S.E.M., *P<0.05) show quantification of these. (D) L6 myotubes were AA-depleted for 1 h in EBSS followed by incubation in EBSS+AA for 15 min in the absence or presence of 50 μM SB415286, 10 μM SB216763, 30 μM roscovitine, 40 μM CT99021 or 100 nM rapamycin for 0.5 h. Cells were harvested and 30 μg of protein was analysed by immunoblotting using the antibodies indicated. (E) HEK293T cells were subjected to AA-depletion/refeeding as in (A–C) or incubated with EBSS supplemented with serum [10% (v/v) FBS] ± 100 nM rapamycin or 50 μM SB415286 for 0.5 h, as indicated. Cells were lysed and 30 μg of protein was analysed by immunoblotting using the antibodies indicated.