Fig. 2

LRRK2 kinase activity regulates LRRK2-PKA RIIβ interaction. a Cell lysates from HEK293T co-transfected with 3xFlag LRRK2 WT and GFP-PKA RIIβ or GFP-empty vector were subjected to co-IP with anti-Flag M2 affinity gel, followed by Flag and GFP immunoblotting. b Pull-down assays of purified GFP-LRRK2 WT or GFP-empty vector incubated with BV2 lysates were subjected to immunoblotting with GFP and PKA RIIβ antibodies. c Cells lysates from HEK293T co-transfected with 3xFlag LRRK2 WT or 3xFlag LRRK2 G2019S and GPF-PKA RIIβ were subjected to co-IP with anti-Flag M2 affinity gel, followed by Flag, GFP, and P-LRRK2 immunoblotting. Quantification LRRK2- RIIβ interaction has been obtained by normalization of RIIβ for LRRK2 protein. Data are representative of three independent experiments (bars represent the mean ± SEM; unpaired t test; *p < 0.05). d Cell lysates from HEK293T co-transfected with 3xFlag LRRK2 WT and GFP-empty vector or 3xFlag LRRK2 WT and GPF- PKA RIIβ treated with GSK, forskolin (Forsk), or DMSO as control (CTR) were subjected to co-IP with anti-Flag M2 affinity gel, followed by Flag and GFP immunoblotting. Quantification LRRK2-RIIβ interaction has been obtained by normalization of RIIβ for LRRK2 protein. Data are representative of four independent experiments (bars represent the mean ± SEM; one-way ANOVA with Tukey’s post-test; **p < 0.01). e Pull-down assays of purified GFP-LRRK2 WT incubated with BV2 lysates previously treated with GSK or DMSO as control (CTR) were subjected to immunoblotting with GFP and PKA RIIβ antibodies. Quantification LRRK2-RIIβ interaction has been obtained by normalization of RIIβ for LRRK2 protein. Data are representative of five independent experiments (bars represent the mean ± SEM; unpaired t test; *p < 0.05)