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

Figure 2. Effects of shRNA-mediated gene silencing of GSK3α/β upon mTORC1-directed signalling.

Figure 2

(A) Total RNA was extracted from L6 myotubes stably expressing either a non-specific shRNA or shRNAs targeting GSK3α and GSK3β. cDNA was synthesized from the mRNA and relative GSK3 mRNA abundance, as measured against GAPDH mRNA, assessed by quantitative PCR. (B) Lysates were prepared from L6 myotubes stably expressing either a non-specific shRNA or shRNAs targeting GSK3α and GSK3β. A 30 μg amount of lysate was analysed by immunoblotting using anti-GSK3α/β or anti-GAPDH antibodies. In addition, 100 μg of lysate was also subjected to immunoprecipitation using antibodies against either GSK3α or GSK3β and analysis of the respective GSK3 activities carried out using a phospho-GS peptide as a substrate. (C) L6 myotubes stably expressing either a non-specific shRNA or shRNAs targeting GSK3α and GSK3β were held in EBSS containing or lacking AA for 1 h or alternatively having been depleted of AAs incubated in EBSS containing a 1× physiological AA mix (refeed) for 15 min in the absence or presence of 50 μM SB415286. Cells were harvested and 30 μg of lysate was analysed by immunoblotting using the antibodies indicated. The asterisk indicates a significant (P<0.05) change between the indicated bars.