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Fig. 4 | Alzheimer's Research & Therapy

Fig. 4

From: Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph

Fig. 4

PLCγ2 protective variant p.P552R shows a slightly hypermorphic activity by increasing intracellular calcium release. a PLC activity of the p.P522R variant under basal and stimulated conditions. Measurement of % IP release in non-transfected COS7 cells (COS7), COS7 cells transfected with pEGFPC1-PLCG2 constructs (common variant, PLCG2 WT; AD associated, rare variant PLCG2 P522R; and PLCG2 D993G (Ali5) mutation. Western blotting was used to confirm equal expression (anti-GFP, inset). SD is represented by error bars. The data are representative for 3 independent experiments. b HEK293T were transfected with mock, EGFR, EGFR and PLCG2 WT, or EGFR and PLCG2 P522R plasmids. c Quantification of IP1 by ELISA IP-One. Transfected HEK293T cells were stimulated with EGF 150 ng/ml and analyzed for IP1 quantification. ELISA reading were averaged of ± standard error (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ####p < 0.0001, two-way ANOVA, Bonferroni multiple comparisons, *compared to control, #compared to EGFR). d Transfected HEK293T cells loaded with Fura-2AM were stimulated with human EGF 150 ng/ml. Intracellular calcium level was recorded by calculating the 340/380 nm excitation ratio at each 5 s. The net intracellular Ca2+ releases value was obtained by subtracting the average of 340/380 excitation ratio values between 0 and 50 s from the maximum 340/380 excitation ratio value between 60 and 200 s, after the EGF stimulation. The net extracellular Ca2+ entry value was obtained by subtracting the average 340/380 excitation ratio values between 285 and 380 s from the average of 340/380 excitation ratio values between 215 and 235 s after adding the extracellular calcium. Data in d are shown as averages of ± standard error (*p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001, & p < 0.05, one-way ANOVA, Tukey’s multiple comparison test, *compared to mock, #compared to EGFR, & compared to PLCγ2. The “n” is corresponding to an average of 45 reading from 3 independent transfection). e Linear representation of the domains of PLCG2 with locations of the point mutations (Y495C, Ali14; P522R, AD protective variant; S707Y, APLAID; D993G, Ali5)

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