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. 2016 Jan 4;113(3):E282–E290. doi: 10.1073/pnas.1516138113

Fig. 4.

Fig. 4.

(A) Intracellular calcium measurements in PMCA2KD cells relative to control. Numbers are mean calcium concentrations estimated by FURA2 measurements. Asterisk denotes statistically significant difference. (B) Co-IP for HER2 and PMCA2 from SKBR3 cells exposed to 2 mM calcium or 10 mM calcium + ionomycin for 16 h. (C) Typical immunoblot for SKBR3 cells exposed to 2 or 10 mM calcium in the absence (−) or presence (+) of ionomycin for 16 h. (D) Confocal images of immunofluorescence for HER2 (green) and phalloidin (actin, red) in control SKBR3 cells exposed to 2 mM calcium (Top) or 10 mM calcium + ionomycin (Bottom) for 16 h. (Insets) Z-stack images in two different orientations; dotted line represents the apical surface of the cell. (E) Costaining for HER2 and EGFR, HER2 and FK2, or HER2 and HER3 in cells exposed to 10 mM calcium + ionomycin in serum-free media (SFM) for 24 h followed by 2 h of EGF. Arrows note intracellular costaining for HER2 and EGFR, HER2 and FK2, or HER2 and HER3. (F) Quantification of cell surface, biotinylated HER2 in control cells exposed to differing concentrations of extracellular calcium ± ionomycin. Values are expressed relative to control conditions at 2 mM calcium. Asterisks denote statistically significant differences compared with control at 2 mM calcium. (G) Diagram illustrating PMCA2T692K mutation. (H) Immunoblot for control T47D cells and T47D cells overexpressing PMCA2T692K or wtPMCA2 in response to doxycycline. (I) Intracellular calcium in T47D cells expressing PMCA2T692K or wtPMCA2 relative to controls (n = 3). Numbers refer to the mean calcium concentrations. Asterisks denote statistically significant differences relative to control cells. (J) BrdU incorporation in T47D cells overexpressing PMCA2T692K or wtPMCA2 relative to controls (n = 3). (K) Apoptosis in control T47D cells and T47D cells overexpressing PMCA2T692K or wtPMCA2 exposed to varying calcium concentrations ± ionomycin relative to control cells at 0.1 mM calcium. Within the same treatment group, * denotes significant differences with PMCA2-T692K; # denotes significant differences with control (n = 3). (Scale bars, 10 μm.)