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. 2021 Nov 19;297(6):101428. doi: 10.1016/j.jbc.2021.101428

Figure 3.

Figure 3

Analysis of the activation status of Flag-RHEB in HeLa cells.A, cell lines expressing Flag-RHEB/WT in a Dox-dependent manner were cultured without or with 1 μg/ml of Dox for 24 h, and anti-Flag immunoprecipitates from the cell lysates were subjected to IP-RP-HPLC analysis. Representative chromatogram of guanine nucleotides bound to Flag-RHEB/WT in HeLa cells (left panel). The relative amounts of guanine nucleotides associated with Flag-RHEB/WT were quantified from the peak areas of GDP and GTP (right panel). The data are the means ± SD from three independent experiments. B, cell lines expressing Flag-RHEB/WT in a Dox-dependent manner were cultured without or with 1 μg/ml of Dox for 24 h, and the cell lysates were subjected to Western blot analysis using the indicated antibodies. C and D, effect of TSC2 knockdown on mTORC1 signaling and the guanine-nucleotide bound state of Flag-RHEB/WT. The cell lines expressing Flag-RHEB/WT in a Dox-dependent manner were transfected with negative control (n.c.) or TSC2 siRNAs and cultured for 48 h, followed by 24 h of culture in the presence of 1 μg/ml of Dox. The cell lysates were subjected to Western blot analysis (C) or IP-RP-HPLC analysis (D). The data are the means ± SD from three independent experiments. ∗ p < 0.05, Student’s t test. Dox, doxycycline; IP-RP-HPLC, ion-pair reversed-phase HPLC; mTORC1, mechanistic target of rapamycin complex 1; RHEB, Ras homolog enriched in brain.