Fig. 2

LRRK2-GS localizes to the ER membrane following dissociation from 14 to 3-3 s. a Upper: Confocal images of endogenous LRRK2 and calnexin in astrocytes isolated from non-Tg and LRRK2-GS mice. Lower: Statistical analysis of areas of overlap between LRRK2 and calnexin in nuclear envelope or peripheral ER regions. Images were analyzed using the Leica Application Suite and ImageJ software. Quantification was performed for 20 randomly selected cells from three independent experiments. b Western blot of the indicated proteins in ER-enriched fractions from non-Tg and LRRK2-GS astrocytes, incubated with or without proteinase K (PK). LRRK2 was detected using antibodies against the C- or N-terminus of LRRK2 (N241A/34 and N138/6, respectively). Calnexin, an integral membrane protein, Bip, an ER lumen protein, and Tom40, an import channel of the mitochondria outer membrane served as controls for the effect of PK. c Immunoprecipitation of LRRK2 in non-Tg and LRRK2-GS astrocytes, followed by Western blotting for the indicated proteins. d Western blot of the indicated proteins in ER fractions from non-Tg and LRRK2-GS astrocytes transfected with siRNAs targeting each 14–3-3 subtype. Band intensities were quantified densitometrically (lower). All graphs represent means ± SD of three independent experiments (*p < 0.05)