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. 2024 Sep 4;15(9):647. doi: 10.1038/s41419-024-07037-2

Fig. 8. The interaction with the TSC1/TSC2 complex is not required for the effects of TBC1D7 on glycolysis in BC cells.

Fig. 8

A, B. HeLa and MDA-MB-468 cells were transfected with 10 nM of siCTRL, siTBC1D7 or siTSC2 (as shown on top). Silenced cells were left in complete medium, growing conditions (GC) (A), or serum starved for 16 h (SF) (B) before harvesting. Total cellular lysates (15 μg) were IB with the antibodies indicated on the right (s.e., short exposure; l.e., long exposure, in these and in the other panels). Vinculin (1), loading control for pS6K and TBC1D7; Vinculin (2), loading control for TSC2 and S6K. MW markers are shown in KDa in these and subsequent panels. C Total cellular lysates (1 mg) from MDA-MB-468 cells, stably expressing empty vector, HA-tagged TBC1D7 wild type (TBC1D7-WT) or TBC1D7-MUT (a TBC1D7 mutant in which Arg81, Gln84 and Arg121 were mutagenized to Ala) were immunoprecipitated with the anti-HA antibody and blotted as indicated on the right. Input, 15 μg of total lysates. D Stable MDA-MB-468 cells expressing the empty vector, HA-tagged TBC1D7-WT or TBC1D7-MUT were silenced as in A, and IB with the antibodies indicated on the right. The red arrow points to endogenous TBC1D7 which is effectively silenced. E MDA-MB-468 cells, treated as in D were assayed for intracellular content of L-lactate. Data are expressed as mean ± SD of l-lactate per cell, normalized to the respective siCTRL in each transfectant, and represent 15 technical replicates from 2 independent experiments. **P < 0.01; ***P < 0.001; ns, not-significant. F MDA-MB-468 cells stably expressing empty vector, HA-tagged TBC1D7-WT or TBC1D7-MUT were analyzed with the indicated antibodies. Vinculin (1), loading control for pS6K and TBC1D7; Vinculin (2), loading control for S6K and HA.