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Fig. 6 | BMC Biology

Fig. 6

From: A two-pore channel protein required for regulating mTORC1 activity on starvation

Fig. 6

The growth and developmental phenotypes of tpc2− cells are dependent on increased mTORC1 activity. a Ax2 or tpc2− cells were grown in shaking suspension as described in the legend of Fig. 5b. Cells were harvested at the times shown. Whole cell lysates were immunoblotted using antibodies against 4E-BP1 phosphorylation (T37/T46). MCCC1 was used as a loading control [41]. One experiment representative of five is shown. The level of 4E-BP1 phosphorylation was quantified with Image Studio Lite (LI-COR), and the average of five independent experiments is shown. b Growth of cells was analysed as described (Fig. 5b) but in the presence of the indicated concentrations of rapamycin or DMSO (vehicle control). The percentage inhibition of cell number is shown, Ax2 in black and tpc2− in grey. Control Ax2 cell number at 72 h is defined as 100%. Average and SEM of four independent experiments. c Exponentially growing parental Ax2 and tpc2− cells were harvested and resuspended at a density of 1.4 × 107 cells/ml in HKC buffer and incubated at 22 °C and 120 rpm shaking for the times shown. Whole cell lysates were immunoblotted using antibodies against phosphorylated 4E-BP1. The level of MCCC1 was used as a loading control [41]. One experiment representative of three. The level of 4E-BP1 phosphorylation was quantified with Image Studio Lite (LI-COR), and the average of three independent experiments is shown ± SEM. Statistical analysis was conducted using Student t tests. Non-significant, NS; P > 0.05; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. d Ax2 and tpc2− cells were developed under buffer as described in the legend of Fig. 2b in the presence of the indicated concentrations of rapamycin or AZD8055. Wells were imaged after 6 h. Scale bars, 200 μm

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