Fig. 2.

Nbs modulate LRRK2 activity in cells. (A) Influence of Nbs on the kinase activity of the LRRK2(G2019S) variant in HEK293T cells. LRRK2 (G2019S) and its effector Rab29 were overexpressed together with GFP-tagged Nbs in HEK293T cells. A negative control, in which no Nb is overexpressed (“No Nb”), is also included. In rows labeled “pLRRK2,” LRRK2 pS1292 levels are determined by Western blot using a site-specific anti-pLRRK2 (pS1292) antibody (Abcam, ab203181) (shown at different times of development). In the rows labeled “pRAB10,” endogenous pT73-Rab10 levels are determined by Western blot using the MJFF (Michael J. Fox Foundation)/Abcam antibody MJF-R21 (Abcam, ab230261) (shown at different times of development). The three lower rows contain controls of LRRK2, GFP-Nb, and Rab10 expression levels, determined on a different Western blot than pLRRK2 and pRab10. Blot is representative of n = 3 (SI Appendix, Fig. S6). (B) Quantification of three different replicates (A and SI Appendix, Fig. S6) based on the densitometric analysis of the Western blots relative to the control (no Nb expressed). (Left) LRRK2 autophosphorylation at S1292. (Right) LRRK2-mediated Rab10 phosphorylation at T73. Median values (bars) and SDs (error bars) are shown. Individual values representing the three replicates are shown as individual datapoints.