pgfp hrab8a q67l nachury  (Addgene inc)

Journal: iScience

Article Title: Pathogenic LRRK2 regulates centrosome cohesion via Rab10/RILPL1-mediated CDK5RAP2 displacement.

doi: 10.1016/j.isci.2022.104476

Figure Lengend Snippet: Figure 2. LRRK2-mediated centrosomal cohesion deficits depend on both GTP conformation and phosphorylation status of Rab8a (A) Example of HEK293T cells co-transfected with FLAG-tagged wild-type LRRK2 and GFP-tagged Rab8a-Q67L (GTP-trapped) or GFP-tagged Rab8a-Q67L- T72A (GTP-trapped but non-phosphorylatable), and stained with antibody against FLAG (red) and the centrosomal marker pericentrin (pseudocolored blue) and DAPI. Arrows point to centrosomes in transfected cells. Cell boundaries (yellow) are shown and were determined by FLAG staining because of FLAG- tagged wild-type LRRK2 expression (or GFP because of GFP-tagged Rab8a expression for single transfection experiments). Scale bar, 10 mm. (B) Quantification of the split centrosome phenotype in cells expressing the indicated constructs, in either the absence or presence of LRRK2 kinase inhibitor MLi2 (100 nM, 2 h) as indicated. Around 100–150 cells were quantified per condition and experiment. Bars represent mean G SEM (n = two to three in- dependent experiments; wt-LRRK2 versus wt-LRRK2 + Rab8a, p = 0.007; wt-LRRK2 + Rab8a versus wt-LRRK2 + Rab8a + MLi2, p = 0.024; wt-LRRK2 versus wt- LRRK2 + Rab8a-Q67L, p = 0.003; wt-LRRK2 + Rab8a-Q67L versus wt-LRRK2 + Rab8a-Q67L + MLi2, p = 0.026); ***p < 0.005; **p < 0.01; *p < 0.05. (C) Cells were co-transfected with FLAG-tagged wild-type LRRK2 and GFP-tagged Rab8a constructs as indicated, left untreated or incubated with 100 nM MLi2 for 2 h, and extracts blotted for FLAG-tagged LRRK2 (FLAG-LRRK2), FLAG-tagged S935-phosphorylated LRRK2 (FLAG-pS935-LRRK2) as a readout for on-target effect of MLi2, phosphorylated GFP-tagged Rab8a (GFP-pT72-Rab8a), total GFP-tagged Rab8a (GFP-Rab8a), and tubulin or GAPDH as loading controls. (D) HeLa cells were transfected with the indicated GFP-tagged Rab8a constructs, briefly fixed, washed and stained with DAPI. All GFP-tagged Rab8a constructs except the inactive T22N mutant display a localization consistent with their presence in a tubular early recycling compartment. Scale bar, 10 mm.

Article Snippet: pDEST53 GFP-hLRRK2 WT Greggio et al., 2006 Addgene Cat#25044 pDEST53 GFP-hLRRK2 G2019S Greggio et al., 2006 Addgene Cat#25045 pDEST53 GFP-hLRRK2 R1441C Greggio et al., 2006 Addgene Cat#25046 pDEST53 GFP-hLRRK2 Y1699C Greggio et al., 2006 Addgene Cat#25048 pDEST53 GFP-hLRRK2 K1906M Blanca Ramı́rez et al., 2017 N/A pCHMWS 3xflag-hLRRK2 Greggio et al., 2009 Prof. Elisa Greggio pCHMWS 3xflag-hLRRK2 Y1699C Greggio et al., 2009 Prof. Elisa Greggio pGFP-hRab8a Nachury et al., 2007 Addgene Cat#24898 pGFP-hRab8A Q67L Nachury et al., 2007 Addgene Cat#24900 pGFP-hRab8a T22N Nachury et al., 2007 Addgene Cat#24899 pGFP-hRab8a T72A Madero-Pérez et al., 2018 N/A pGFP-hRab8A Q67L-T72A This study N/A GFP-hTBC1D30 Yoshimura et al., 2007 Prof. Francis Barr GFP-hTBC1D4 Yoshimura et al., 2007 Prof. Francis Barr GFP-hTBC1D4 R973K Yoshimura et al., 2007 Prof. Francis Barr GFP-hTBC1D17 Yoshimura et al., 2007 Prof. Francis Barr (Continued on next page) 18 iScience 25, 104476, June 17, 2022

Techniques: Phospho-proteomics, Transfection, Staining, Marker, Expressing, Construct, Incubation, Mutagenesis