Fig. 3. LRRK2 interacts with Beclin-1 and suppresses autophagy.

(A and B) BMDCs from Lrrk2 Tg mice (Lrrk2 Tg) or littermate control mice were cultured with or without BafA1 (300 nM) for 30 min and then were stimulated with M. leprae for 2 hours. The cell lysates were subjected to immunoblotting to examine autophagic flux. Total RNA was extracted from BMDCs, and p62 mRNA expression was determined using real-time PCR. (B) Lrrk2 Tg mice, n = 3; control mice, n = 3; P values from left to right: P = 0.6819, P = 0.9586, P = 0.3713, and P = 0.3073). NS, not significant. (C) BMDCs from Lrrk2 Tg mice and littermate control mice that had been crossed with GFP-LC3 Tg mice were stimulated with M. leprae and then were evaluated for the number of GFP-LC3 puncta by confocal laser scanning microscopy. The data shown consist of pooled data from three independent experiments. (C) Lrrk2 Tg mice, n = 3; control mice, n = 3; M. leprae: 6 hours, P = 0.0010; M. leprae: 16 hours, P < 0.0001. *P < 0.05 was considered a statistically significant difference. Statistical significance was determined using a Student’s t test. (D) BMDCs from Lrrk2 Tg mice were stimulated with HK-CA for 60 min in culture and then were stained with anti-FLAG antibody (green) and anti-LAMP1 antibody (red). DIC, differential interference contrast. (E) Whole-cell lysates of HEK293T cells cotransfected with GFP-LRRK2 and FLAG–Beclin-1 plasmids were subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-GFP antibody. (F) BMDCs from Lrrk2 Tg mice cultured with or without HK-CA were subjected to a proximity ligation assay (PLA) to detect LRRK2/Beclin-1 complexes (red). PLA-, no PLA probe, negative control; DAPI, 4′,6-diamidino-2-phenylindole.