Figure S1. Validation of... | Rockefeller University Press

Figure S1.

Validation of LRRK2-expressing SH-SY5Y cell lines and effects of ROC/COR and kinase-inactivating mutations on cytosolic Ca2+. (a) Western blot analyses using an antibody to LRRK2 in the parental SH-SY5Y cell line and cell lines expressing EV, wild-type LRRK2 (WT), LRRK2 G2019S (GS), KI LRRK2 (KI), LRRK2 R1441C (RC), and LRRK2 R1441G (RG). Expression of wild-type and G2019S LRRK2 in published lines (102 and 103, respectively)52 was processed in parallel. Blots were reprobed with an antibody to cathepsin D (CTSD). (b) Quantitative PCR analysis of LRRK2 in the various cell lines used in this study. Data (mean ± SD, n = 3 technical replicates) are normalized to the expression of ubiquitin. (c) Schematic of LRRK2 showing the position of the three mutations introduced to generate the KI construct and the two individual ROC/COR mutations (RG/RC). (d) Exemplar Ca2+ signals recorded from cells loaded with Fura-2 from the lines expressing KI LRRK2. Gray lines are responses from individual cells. The thick lines are the population average. (e) Ca2+ signals from multiple population averages (mean ± SEM). n = 6 (KI 1), n = 5 (KI 4), where n refers to the number of independent biological replicates. (f) Schematic of LRRK2 showing the position of the two individual ROC/COR mutations (RG/RC). (g) Exemplar Ca2+ signals recorded from cells loaded with Fura-2 from the lines expressing the ROC/COR LRRK2 mutants. Gray lines are responses from individual cells. The thick lines are the population average. (h) Ca2+ signals from multiple population averages (mean ± SEM). n = 5 (RC 4), n = 8 (RG 5), where n refers to the number of independent biological replicates. (i) Summary data quantifying the peak change and the area under the curve for the Ca2+ signals. Data are presented as the mean ± SEM and amalgamated for both lines expressing KI LRRK2 and the ROC/COR mutants where each point is an independent biological replicate. Data are compared with amalgamated control lines (parental and empty vector). **P < 0.01 (Kruskal–Wallis, Dunn’s test), ****P < 0.0001 (one-way ANOVA, Tukey’s test). WT, wild type; EV, empty vector; KI, kinase inactive. Source data are available for this figure: SourceData FS1.

Validation of LRRK2-expressing SH-SY5Y cell lines and effects of ROC/COR and kinase-inactivating mutations on cytosolic Ca ²⁺ . (a) Western blot analyses using an antibody to LRRK2 in the parental SH-SY5Y cell line and cell lines expressing EV, wild-type LRRK2 (WT), LRRK2 G2019S (GS), KI LRRK2 (KI), LRRK2 R1441C (RC), and LRRK2 R1441G (RG). Expression of wild-type and G2019S LRRK2 in published lines (102 and 103, respectively)⁵² was processed in parallel. Blots were reprobed with an antibody to cathepsin D (CTSD). (b) Quantitative PCR analysis of LRRK2 in the various cell lines used in this study. Data (mean ± SD, n = 3 technical replicates) are normalized to the expression of ubiquitin. (c) Schematic of LRRK2 showing the position of the three mutations introduced to generate the KI construct and the two individual ROC/COR mutations (RG/RC). (d) Exemplar Ca²⁺ signals recorded from cells loaded with Fura-2 from the lines expressing KI LRRK2. Gray lines are responses from individual cells. The thick lines are the population average. (e) Ca²⁺ signals from multiple population averages (mean ± SEM). n = 6 (KI 1), n = 5 (KI 4), where n refers to the number of independent biological replicates. (f) Schematic of LRRK2 showing the position of the two individual ROC/COR mutations (RG/RC). (g) Exemplar Ca²⁺ signals recorded from cells loaded with Fura-2 from the lines expressing the ROC/COR LRRK2 mutants. Gray lines are responses from individual cells. The thick lines are the population average. (h) Ca²⁺ signals from multiple population averages (mean ± SEM). n = 5 (RC 4), n = 8 (RG 5), where n refers to the number of independent biological replicates. (i) Summary data quantifying the peak change and the area under the curve for the Ca²⁺ signals. Data are presented as the mean ± SEM and amalgamated for both lines expressing KI LRRK2 and the ROC/COR mutants where each point is an independent biological replicate. Data are compared with amalgamated control lines (parental and empty vector). **P < 0.01 (Kruskal–Wallis, Dunn’s test), ****P < 0.0001 (one-way ANOVA, Tukey’s test). WT, wild type; EV, empty vector; KI, kinase inactive. Source data are available for this figure: SourceData FS1.

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