Figure 2.

NCLX activity is controlled by phosphorylation on Ser271. (A) Representative immunoblot analysis (Western blot) of NCLX c-myc-tagged constructs (For Antibodies see Table 1). Extracts of HEK293 cells transfected with shNCLX and either the NCLX^WT, NCLX^S271A, NCLX^S271D or control vector pcDNA3.1+ (vector) were subjected to immunoblot analysis with c-Myc and β-Actin antibodies. (B) Densitometry analysis of the Western blot shown in (A). Protein expression was normalized to β-Actin expression (unpaired two-tailed t-test, NCLX^WT vs. Vector: t = 3.979, df = 4, *p = 0.0164; NCLX^WT vs. NCLX^S271A: t = 1.469, df = 4ns; NCLX^WT vs. NCLX^S271D: t = 1.055, df = 4, ns; n = 3). (C) Representative fluorescent traces of ATP (100 μM) induced [Ca²⁺]m transients in Cepia2mt expressing SH-SY5Y cells co-expressing shNCLX and NCLX^WT (black) or mutant NCLX^S271A (purple)\NCLX^S271D (turquoise). (D) Quantification of [Ca²⁺]m influx amplitude in (C) (Welch and Brown-Forsythe ANOVA, F(2,78.46) = 5.028; Dunnett’s T3 multiple comparisons test; NCLX^WT vs. NCLX^S271A: *p = 0.011; NCLX^WT vs. NCLX^S271D: ns; n > 3). (E) Quantification of [Ca²⁺]m efflux rates in (C) (Welch and Brown-Forsythe ANOVA, F(2,77.72) = 5.519; Dunnett’s T3 multiple comparisons test; NCLX^WT vs. NCLX^S271A: **p = 0.0091; NCLX^WT vs. NCLX^S271D: ns; n > 3). (F,G) Representative fluorescent traces of ATP (100μM) induced [Ca²⁺]m transients in Cepia2mt expressing SH-SY5Y cells co-expressing shNCLX and NCLX^WT (black or grey with TBCA) or mutant NCLX^S271A (purple or pink with TBCA)\NCLX^S271D (turquoise or light blue with TBCA) treated with TBCA. (H) Quantification of [Ca²⁺]m efflux rates in (G,H) (Welch and Brown-Forsythe ANOVA, F(5,220.7) = 7.435; Dunnett’s T3 multiple comparisons test; NCLX^WT vs. NCLX^WT+TBCA: ***p = 0.0006; NCLX^WT vs. NCLX^S271A: *p = 0.0126; NCLX^WT vs. NCLX^S271A+TBCA: *p = 0.0292; NCLX^WT vs. NCLX^S271D: ns; NCLX^WT vs. NCLX^S271D+TBCA: ns; n > 3). ns—not significant. Error bars denote SEM.