Journal: bioRxiv

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1101/2024.08.19.608556

Figure Lengend Snippet: (A) HEK293 cells were cultured in serum-starved condition (−FBS) for 3 h and then stimulated with or without (-) carbachol (100 μM) for 15 or 30 min (+CCh). Cell lysates were analyzed by Western blotting with the indicated antibodies. (B) HEK293 cells transiently transfected with the plasmid encoding R-GECO1 were deprived of serum for 3 h, then carbachol (100 μM) were added at 120 s. Ratio of fluorescence signals for R-GECO1 to the average signals before carbachol stimulation (F 0 ) were shown. Graphs are shown as mean ± SD (n = 12). (C) HEK293 cells were cultured in serum-starved condition (−FBS) for 3 h pretreated with DMSO or BAPTA-AM (50 µM) for the last 1 h and then stimulated with or without (-) carbachol (100 μM) for 15min (+CCh). Cell lysates were analyzed by Western blotting with the indicated antibodies. (D) Quantitation of the relative intensity of phospho-T389-S6K1 to total S6K1 of (C), in the carbachol stimulation (DMSO) was set to 1. Graphs represent mean ± SEM of three independent experiments, One-way ANOVA with Tukey’s test, * p < 0.05, *** p < 0.001. (E) HEK293 cells were cultured in serum-starved condition (−FBS) for 3 h pretreated with DMSO or CaM inhibitors (W-7 (30 µM) or Calmidazolium (CMDZ, 30 μM)) for the last 15 min (CMDZ) or 5 min (W-7) and then stimulated with or without (-) carbachol (100 μM) for 15min (+CCh). Cell lysates were analyzed by Western blotting with the indicated antibodies. (F) Quantitation of the relative intensity of phospho-T389-S6K1 to total S6K1 of (E), in the carbachol stimulation (DMSO) was set to 1. Graphs represent mean ± SEM of three independent experiments, One-way ANOVA with Tukey’s test, *** p < 0.001.

Article Snippet: BAPTA-AM solution (348–05451) was purchased from Wako (Tokyo, Japan).

Techniques: Cell Culture, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Quantitation Assay

Journal: bioRxiv

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1101/2024.08.19.608556

Figure Lengend Snippet: (A) HEK293 cells were starved of serum (−FBS) for 3 h, pretreated with DMSO or BAPTA-AM (50 µM) or W-7 (30 μM) for the last 1 h (BAPTA-AM) or 5 min (W-7) and then stimulated with or without (-) carbachol (100 μM) for 15 min (+CCh). Cell lysates were analyzed by Western blotting with the indicated antibodies. (B) Quantitation of the relative intensity of phospho-T1462-TSC2 to total TSC2 of (A), in which the carbachol stimulation (DMSO) was set to 1. Graphs represent mean ± SEM of three independent experiments. One-way ANOVA with Tukey’s test. (C) HEK293 cells were starved of serum (-FBS) for 3 h, and then treated with carbachol (100 μM) or insulin (100 nM) for 15 min. The cells were then immunostained with anti-TSC2 and anti-LAMP2 antibodies. Merged images of TSC2 (green), LAMP2 (magenta) and nuclei staining with Hoechst 33342 (blue) are also shown. Scale bar, 10 µm. (D) Colocalizations of TSC2 with LAMP2 were quantified, and Pearson’s correlation coefficient is shown. n = 20. One-way ANOVA with Tukey’s test, * p < 0.05, *** p < 0.001.

Article Snippet: BAPTA-AM solution (348–05451) was purchased from Wako (Tokyo, Japan).

Techniques: Western Blot, Quantitation Assay, Staining