Fig. 3.

Calcium deficiency suppresses mTORC1 activity, activating TFEB. A Immunoblot analysis of AML12 cells in calcium-deficient medium for indicated durations. Whole cell lysates were reacted with anti-phosphoS6, anti-S6, and anti-β-actin. B Cytosolic and nuclear fractions from AML12 cells with or without calcium for 24 h. Immunoblot fractions with anti-TFEB and p-TFEB (Ser211). For the cytosolic and nuclear fractions, alpha-tubulin and CREB antibodies were utilized as the controls for cytosolic and nuclear fractionations, respectively. C Immunofluorescence of AML12 cells with or without calcium with anti-TFEB (red). Scale bar represents 25 µm. D Quantification of red fluorescence in the nucleus indicates activation of TFEB. Data are expressed as means ± S.D. (n = 3, independent experiments, 30 cells were analyzed in each experiment), * p < 0.05