Additional information regarding RILPL1 recruitment to lysosomes by LRRK2. (A) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded for 24 h and treated with DMSO, LLOME (2 h), and LLOME+MLi2. Cells were then fixed and stained for HA and endogenous RILPL1. (B) Mouse primary astrocytes were transfected with 3xflag-LRRK2 for 48 h. Cells were then treated or not with LLOME for 6 h, fixed, and stained for endogenous LAMP1 and RILPL1. (C) U2OS cells were treated with a nontargeting control (NTC) or siRILPL1 for 60 h. Cells were then lysed and blotted for endogenous RILPL1. (D) U2OS cells were treated with a NTC or siRILPL1 for 24 h. Cells were then transfected with 3xflag-LRRK2 for 36 h, treated with LLOME for 2 h, and fixed. Cells were stained for endogenous RILPL1 and LAMP1. Graph shows the number of RILPL1-positive lysosomes per cell in both conditions (n = 13–14 cells). (E) U2OS cells were transfected with LAMP1-HaloTag and 3xflag-LRRK2 (NT-LRRK2) or LAMTOR1(1–39)-3xflag-LRRK2 (LYSO-LRRK2) for 36 h. Cells were fixed and stained for endogenous RILPL1. (F) U2OS cells were transfected with HaloTag-LRRK2 and mCherry-RILPL1 for 36 h. Cells were then treated or not with LLOME (2 h) or pre-treated with MLi2. Lysates were subjected to immunoprecipitation with anti-RFP antibodies. (G) HEK293T cells were transfected with 3xflag-LRRK2 and 2xmyc-RILPL1 for 24 h. Then cells were treated or not with LLOME for 2 h. Lysates were subjected to immunoprecipitation with anti-myc antibodies and blotted against pS106-RAB12 and myc antibodies. (H) HEK293T cells were transfected with 3xflag-LRRK2 and 2xmyc-RILPL1 for 24 h. Then cells were treated or not with LLOME for 2 h. Lysates were subjected to immunoprecipitation with anti-myc antibodies and blotted against JIP4, pT73-RAB10, and myc antibodies. (I) U2OS cells were treated with a NTC or siRILPL1 for 24 h. Cells were then transfected with LifeAct-mNeonGreen for 36 h, treated or not with LLOME for 2 h, and fixed. Cells were then stained for endogenous LAMP1. (J and K) Graphs show the perinuclear ratio of lysosomes in the different conditions. Unpaired t test. Data are mean ± SEM (n = 20 cells). (L) U2OS cells transfected with HaloTag-LRRK2 and mNeonGreen-JIP4 for 36 h and treated with LLOME for 2 h before fixing. Cells were then stained for endogenous RILPL1. Scale bar = 10 µm. Source data are available for this figure: SourceData FS3.
Figure S3.

Additional information regarding RILPL1 recruitment to lysosomes by LRRK2. (A) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded for 24 h and treated with DMSO, LLOME (2 h), and LLOME+MLi2. Cells were then fixed and stained for HA and endogenous RILPL1. (B) Mouse primary astrocytes were transfected with 3xflag-LRRK2 for 48 h. Cells were then treated or not with LLOME for 6 h, fixed, and stained for endogenous LAMP1 and RILPL1. (C) U2OS cells were treated with a nontargeting control (NTC) or siRILPL1 for 60 h. Cells were then lysed and blotted for endogenous RILPL1. (D) U2OS cells were treated with a NTC or siRILPL1 for 24 h. Cells were then transfected with 3xflag-LRRK2 for 36 h, treated with LLOME for 2 h, and fixed. Cells were stained for endogenous RILPL1 and LAMP1. Graph shows the number of RILPL1-positive lysosomes per cell in both conditions (n = 13–14 cells). (E) U2OS cells were transfected with LAMP1-HaloTag and 3xflag-LRRK2 (NT-LRRK2) or LAMTOR1(1–39)-3xflag-LRRK2 (LYSO-LRRK2) for 36 h. Cells were fixed and stained for endogenous RILPL1. (F) U2OS cells were transfected with HaloTag-LRRK2 and mCherry-RILPL1 for 36 h. Cells were then treated or not with LLOME (2 h) or pre-treated with MLi2. Lysates were subjected to immunoprecipitation with anti-RFP antibodies. (G) HEK293T cells were transfected with 3xflag-LRRK2 and 2xmyc-RILPL1 for 24 h. Then cells were treated or not with LLOME for 2 h. Lysates were subjected to immunoprecipitation with anti-myc antibodies and blotted against pS106-RAB12 and myc antibodies. (H) HEK293T cells were transfected with 3xflag-LRRK2 and 2xmyc-RILPL1 for 24 h. Then cells were treated or not with LLOME for 2 h. Lysates were subjected to immunoprecipitation with anti-myc antibodies and blotted against JIP4, pT73-RAB10, and myc antibodies. (I) U2OS cells were treated with a NTC or siRILPL1 for 24 h. Cells were then transfected with LifeAct-mNeonGreen for 36 h, treated or not with LLOME for 2 h, and fixed. Cells were then stained for endogenous LAMP1. (J and K) Graphs show the perinuclear ratio of lysosomes in the different conditions. Unpaired t test. Data are mean ± SEM (n = 20 cells). (L) U2OS cells transfected with HaloTag-LRRK2 and mNeonGreen-JIP4 for 36 h and treated with LLOME for 2 h before fixing. Cells were then stained for endogenous RILPL1. Scale bar = 10 µm. Source data are available for this figure: SourceData FS3.

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