1. Knock‐in mutation of the T1348N mutation that ablates GTP binding to LRRK2 abolishes Rab10 and biomarker phosphorylation
Knock‐in mutation of the T1348N mutation that ablates GTP binding to LRRK2 abolishes Rab10 and biomarker phosphorylation HEK293 cells were transfected with the indicated wild‐type and mutant forms of LRRK2 with either HA‐empty vector (−) or HA‐tagged Rab29 (+). 24 h post‐transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies WT is wild type. Similar results were obtained in two experiments.Wild‐type LRRK2, heterozygous LRRK2[T1348N/+], and homozygous LRRK2[T1348N/T1348N] knock‐in MEFs derived from littermate embryos were lysed immunoblotted with the indicated antibodies. Similar results were obtained in two experiments. WT is Wild type and TN corresponds to T1348N.HeLa cells were transfected with FLAG‐T1348N‐LRRK2. After 48 h, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol, then fixed, and stained with rabbit anti‐LRRK2 antibody. Nuclear DAPI stain (blue); LRRK2 (red). Scale bar, 10 μm. Dotted line represents cell outlines.HEK293T cells were transfected with FLAG‐T1348N‐LRRK2 and 24 h later with Myc‐Rab29. After 24 h of Rab29 expression, cytosol and membrane fractions were prepared. Immunoblot of membrane protein (50 μg) and cytosolic protein (40% of equivalent volume) ± Rab29 as indicated. Numbers at left indicate mobility of marker proteins in kDa; proteins were detected with rabbit anti‐LRRK2, mouse anti‐tubulin, and mouse anti‐Myc antibodies. Source data are available online for this figure.
Elena Purlyte et al. EMBO J. 2018;37:1-18
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