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. 2015 May 22;6:7250. doi: 10.1038/ncomms8250

Figure 3. LAPTM4b stimulates mTORC1 activation.

Figure 3

(a,b) LAPTM4b enhances mTORC1 activation: HeLa cells stably transfected with control knockdown (control KD), LAPTM4b knockdown (LAPTM4 KD, targeting its 3′untranslated region) or LAPLTM4b KD reconstituted with mCh-LAPTM4b, were serum- and nutrient-starved and tested for activation of S6K1 (pp70) following (or not) 15 min stimulation with essential amino acids (EAAs). (a) A representative immunoblot. (b) Quantification of mTORC1 activation (pp70/p70 ratio) by LAPTM4b (data are mean±s.e.m., N=3). (c–f) Time course of activation of mTORC1 by LAPTM4b determined by S6K1 (pp70; c,d) or 4E-BP (p4E-BP; e,f) phosphorylation in HeLa cells stably expressing control KD, LAPTM4b KD or LAPTM4b KD reconstituted with mCh-LAPTM4b (LAPTM4b KD+LAPTM4b), serum- and nutrient-starved and stimulated with EAA for the indicated times. DMEM: control (non-starved cells). (c,e) Representative immunoblots. (d,f) Quantification of p70 and 4E-BP phosphorylation (activation), respectively. (g) mTORC1 activation (S6K1 phosphorylation) in serum- and nutrient-starved cells by Leu (0.4 mM, 15 min) alone. The experiment was performed as in a. (h) Rescue of mTORC1 activation in cells knocked down for LAPTM4b and expressing a constitutively active RagA, RagA(Q66L). (g,h) Quantification of data is depicted underneath the immunoblots. For all panels depicting quantification: values are mean±s.e.m. (N=3 independent experiments). P values were calculated from Student's t-tests.