Fig 2: PMCA mRNA levels in MDA-MB-231 breast cancer cells and silencing of PMCA isoforms in MDA-MB-231 cells.(A) PMCA isoform mRNA levels in confluent MDA-MB-231 cells relative to PMCA2 mRNA. (B) PMCA2 mRNA levels in sub-confluent (sub-con.) and confluent (con.) MDA-MB-231 cells. (C) PMCA2, PMCA1 and PMCA4 siRNA mediated silencing, 120 h after transfection with non-targeting siRNA (siNT), PMCA2 siRNA (siPMCA2), PMCA1 siRNA (siPMCA1) or PMCA4 siRNA (siPMCA4). The data are mean ± SD (n = 3) and are from three independent experiments, *P < 0.05, unpaired t-test.

Fig 3: Enhanced interactions between HER2 and MAL2, Ezrin, NHERF1, and PMCA2 in trastuzumab-resistant SKBR3 cells(A) Immunofluorescence staining for cholera toxin B (lipid rafts) in control and trastuzumab-resistant SKBR3 cells. Scale bars represent 10 µm.(B) Immunofluorescence staining for HER2 and MAL2 in control and trastuzumab-resistant SKBR3 cells. Scale bars represent 10 µm.(C) PLA for HER2 and MAL2 in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Scale bars represent 10 µm.(D–F) Quantitative results from immunoprecipitation coupled with data-independent acquisition mass spectrometry (DIA-MS) in control and trastuzumab-resistant SKBR3 cells. (D) The DIA-MS Intensity (log2) of HER2 and MAL2 proteins from control and trastuzumab-resistant SKBR3 cells. (E) The DIA-MS Intensity (log2) for all the peptide precursor signals of HER2 in control and resistant cells. (F) The DIA-MS peak groups visualized for quantifying HER2 (VLGSGAFGTVYK) and MAL2 (VTLPAGPDILR). Peaks above and below the middle line denote the MS2 and MS1 ion traces in DIA-MS.(G) MAL2, Ezrin, and NHERF1 mRNA expression in control and trastuzumab-resistant SKBR3 cells as assessed by quantitative RT-PCR (n = 3).(H) PLA for HER2 with Ezrin (left), NHERF1 (middle), and PMCA2 (right) in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Boxed portions are amplified at right with co-registration of PLA signal and immunofluorescence for actin (phalloidin).(I) Quantitation of PLA experiment for HER2 in combination with MAL2, Ezrin, NHERF1, or PMCA2 represented as the fluorescent intensity of amplified PLA signals associated with membrane protrusions.(J) Coimmunoprecipitation for HER2 and HSP90 in control and trastuzumab-resistant SKBR3 cells.(K) PLA for HER2 and HSP90 in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Scale bars represent 10 µm.(L) XTT cell viability assay in control, MAL2KD-treated, and MßCD-treated trastuzumab-resistant SKBR3 and BT474 cells.(M) Immunofluorescence staining for FOXO1 in control, MAL2KD-treated, and MßCD-treated trastuzumab-resistant SKBR3 cells.(N) Immunofluorescence staining for HER2 and pAKT in control, MAL2KD-treated, and MßCD-treated trastuzumab-resistant SKBR3 cells. Scale bars represent 10 µm.(O) Diagram representing the structure of MAL2- and lipid raft-enriched membrane protrusions containing multi-protein HER2 signaling complexes. These results are representative of three independent experiments.

Fig 4: Effect of overexpression of Atoh1 and Egr1 on endogenous levels of Atp2b2 and Atp2b4. a, b EGR1 increases expression of Atp2b2 in both OC-1 and N2A cell lines. ATOH1 moderately inhibits expression of Atp2b2 in OC-1 (ns) and has no effect in N2A cells. c, d Atp2b4 transcript expression is unaffected by overexpression of EGR1 in both OC-1 and N2A cells. ATOH1 increases expression of Atp2b4 transcript in OC-1 cells but not in N2A cells. Comparisons to empty vector (PIE) were made using a Student’s t test (*P = 0.05, **P = 0.01). Data shown is the average of three technical replicates for three biological replicates in each cell line, variation is shown as standard error of the mean

Fig 5: Expression and immunolocalization of recombinant PMCA2w/a pumps in CHO cells. A, Western blotting analysis is shown. The band of ~130 kDa corresponds to PMCA2, and the band of ~42 kDa corresponds to ß-actin. B, densitometric analysis of relative expression of wild type PMCA2w/a and PMCA2w/a harboring the Tommy mutation is shown. The amounts of pump protein were as normalized for ß-actin. C, immunolocalization of wild type (left) and mutant (right) PMCA2w/a in transiently transfected CHO cells is shown. The interaction with the 2N antibody was probed by the AlexaFluor488-conjugated secondary antibody. D, fluorescence levels in the plasma membrane is quantified as described under “Experimental Procedures.” Bars represent S.D.