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

Figure 5. LAPTM4b requires active V-ATPase for mTORC1 activation.

Figure 5

(a) HeLa cells expressing control KD, LAPTM4b KD or LAPTM4 KD reconstituted with HA-LAPTM4b (LAPTM4b KD+LAPTM4b) were serum- and nutrient-starved overnight and stimulated with EAA for 15 min in the presence/absence of the V-ATPase inhibitor Concanamycin A (ccA, 5 μM). mTORC1 activation was determined by immunoblotting for activated S6K1 (pp70), as in Fig. 3 above. The top panel depicts a representative experiment, while the bottom panel depicts the quantification of three separate experiments. (b) HeLa cells expressing control KD, LAPTM4b KD or LAPTM4 KD reconstituted with HA-LAPTM4b (LAPTM4b KD+LAPTM4b) were transfected with V-ATPase shRNA (ATP6VOc shRNA) to yield 60% knockdown. Cells were serum- and nutrient-starved overnight, stimulated with EAA for 15 min and analysed for S6K1 activation as described in a. In b, the top panel depicts a representative experiment, while the bottom panel depicts the quantification of three separate experiments. In a and b, values are mean±s.e.m. (N=3). P values were calculated from Student's t-tests. NS, not significant.