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. 2023 Oct 24;12:e87255. doi: 10.7554/eLife.87255

Figure 2. Rab12 regulates LRRK2 activation basally and in response to lysosomal damage and genetic variants associated with Parkinson’s disease (PD).

(A) The levels of Rab12, pS106 Rab12, pT73 Rab10, and Rab10 were assessed in wildtype (WT), RAB12 KO, and LRRK2 KO A549 cells by western blot analysis. Shown is a representative immunoblot with GAPDH as a loading control. (B) The levels of pT73 Rab10 were measured using a Meso Scale Discovery (MSD)-based assay. The MSD signal was normalized for protein input and expressed as a fold change compared to WT A549 cells; data are shown as the mean ± SEM; n=4 independent experiments, and statistical significance was determined using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. (C) Immunoblot signals from multiple experiments were quantified, and the Rab10 signal was normalized to GAPDH and expressed as a fold change compared to WT A549 cells. Data are shown as the mean ± SEM; n=4 independent experiments. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparison test. (D–F) RAB12 KO A549 cells with doxycycline-inducible expression of WT RAB12 or a phospho-deficient variant of RAB12 (S106A) were treated with increasing concentrations of doxycycline for 3 days, and the levels of Rab12, pS106 Rab12, pT73 Rab10, and LRRK2 were measured. (D) A representative immunoblot is shown assessing Rab12 and pS106 Rab12 protein levels following doxycycline-induced expression of WT or RAB12 S106A, and GAPDH was used as a loading control. (E and F) The levels of pT73 Rab10 and LRRK2 were measured using MSD-based assays. MSD signals were normalized for protein concentration, and data were then normalized to the median within each batch and to the signals from the control group (RAB12 KO cells with inducible expression of WT Rab12 without doxycycline treatment). Data are shown as mean ± SEM; n=3–4 independent experiments, and statistical significance was determined using unpaired t-test on log transformed data. (G) The impact of Rab12 knockdown was measured in WT, LRRK2 R1441G KI, and VPS35 D620N KI A549 cells. Cells were transfected with siRNA targeting RAB12, and pT73 Rab10 levels were measured by MSD-based analysis 3 days after transfection. The MSD signal was normalized for protein input and then normalized to the median within each batch and is expressed as a fold change compared to WT A549 cells transfected with scramble siRNA. Data are shown as the mean ± SEM; n=5 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison test on log transformed data. (H) WT, RAB12 KO, and LRRK2 KO A549 cells were treated with vehicle or L-leucyl-L-leucine methyl ester (LLOMe) (1 mM) for 2 hr, and the impact of LLOMe treatment on pT73 Rab10 levels was measured by MSD-based analysis. The MSD signal was normalized for protein input and is expressed as a fold change compared to WT A549 cells treated with vehicle. Data are shown as the mean ± SEM; n=4 independent experiments. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2—source data 1. Raw data files for western blot.
Figure 2—source data 2. Annotated western blots.

Figure 2.

Figure 2—figure supplement 1. Confirmation of effects of RAB12 KO and RAB12 S106A KI on total and phospho-Rab12 and Rab10.

Figure 2—figure supplement 1.

(A) The levels of Rab12 were measured in cell lysate from wildtype (WT) or three clones of RAB12 KO A549 cells by western blot analysis. The Rab12 signal was quantified and normalized to the GAPDH signal and expressed as a fold change compared to WT cells. Data are shown as the mean ± SEM; n=4 independent experiments. Statistical significance was determined using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test. (B) The levels of pT73 Rab10 were measured in cell lysates from WT, three clones of RAB12 KO A549 cells, or LRRK2 KO A549 cells by meso scale discovery (MSD)-based analysis and were normalized to the levels of Rab10 (measured by western blot analysis). Data are shown as the mean ± SEM; n=4 independent experiments. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test. (C–G) The levels of pS106 Rab12, Rab12, pT73 Rab10, and Rab10 were measured in cell lysates from WT, three clones of RAB12 S106A KI A549, or RAB12 KO cells by western blot analysis, and GAPDH was used as a loading control. The pS106 Rab12 signal (C), Rab12 signal (D), pT73 Rab10 signal (E), and Rab10 signal (F) were measured and normalized to the GAPDH signal and expressed as a fold change compared to WT cells. pT73 Rab10 levels (measured by MSD-based assay) were normalized to total Rab10 levels (measured by western blot) and expressed as a fold change compared to WT cells (G). Data are shown as the mean ± SEM; n=4 independent experiments. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparison test. (H) Total LRRK2 levels were reduced in LRRK2 R1441G cells compared to WT cells. LRRK2 levels were measured in cell lysates from WT and LRRK2 R1441G KI A549 cells treated with scramble siRNA or siRNA against RAB12 by MSD-based analysis and normalized for protein input. Data are shown as the mean ± SEM; n=5 independent experiments. **p<0.01, ***p<0.001, ****p<0.0001.
Figure 2—figure supplement 1—source data 1. Raw data files for western blot.
Figure 2—figure supplement 1—source data 2. Annotated western blots.