Fig 2: Characterization of MJFF transgenic Rab29 overexpressing mouse.(A) Schematic overview of design and the targeting strategy utilized to create the Rab29 transgenic mouse model. The illustration depicts the transgene containing a neo cassette, a Pgk promoter, the murine Rab29 open reading frame together with a Kozak sequence (GCCACC), the human growth hormone polyadenylation signal and an additional polyadenylation signal. These elements were inserted into the ROSA26 locus via recombinase-mediated cassette exchange. (B) The indicated tissues were collected from 3.5-month-old and 6-month-old wildtype (WT) and homozygous, transgenic Rab29 overexpressing (Tg Rab29) mice. Thirty micrograms of tissue extracts were subjected to quantitative immunoblot analysis with the indicated antibodies. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Quantified data are presented as mean ± SD of total Rab29/GAPDH ratios, calculated using the Image Studio software. Values were normalized to the average of the 3.5-month-old wildtype mice. Each lane represents a tissue sample from a different animal. (C) The indicated tissue extracts from 3.5-month-old wildtype (WT), heterozygous (Tg Rab29 Het), and homozygous (Tg Rab29 Hom) transgenic Rab29 overexpressing mice were processed for total RNA extraction, cDNA synthesis, and subsequent quantitative real-time RT-PCR analysis. Rab29 mRNA levels were normalized to ß-actin mRNA levels and further normalized to the wildtype of the appropriate tissue. Quantified data are presented as mean ± SD of four biological replicates per genotype.

Fig 3: Detection of endogenous Rab29 in human and mouse extracts with novel Rab29 monoclonal antibodies.(A) Thirty micrograms of the indicated cell and tissue extracts were subjected to quantitative immunoblot analysis with the indicated antibodies, diluted to 1 µg/ml. Membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. (B) Various cell and tissue extracts were analyzed for Rab29 and LRRK2 expression (upper panels). Total protein levels were visualized using the Bio-Rad Oriole Fluorescent Gel Stain and exposed using the Bio-Rad Chemidoc MP Imaging System (lower panel). Total protein levels were quantified using Bio-Rad Image Lab software. Total Rab29 or total LRRK2 was quantified using the Image Studio software. Quantified data are presented as ratios of Rab29 or LRRK2 expression divided by total protein levels, and values were normalized to the average of the respective protein expression observed in MEFs. Quantifications are representative of three independent experiments and shown as mean ± SD.

Fig 4: Knock-out of Rab29 does not affect basal LRRK2 activity.(A) Two independent littermate-matched wildtype (WT), Rab29 knock-out heterozygous (+/-), or Rab29 knock-out homozygous (-/-) MEFs were treated with vehicle (DMSO) or 100 nM LRRK2 inhibitor MLi-2 for 90 min prior to harvest. Twenty micrograms of whole-cell extracts were subjected to quantitative immunoblot analysis with the indicated antibodies. Technical replicates represent cell extract obtained from a different dish of cells. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Quantified data are presented as the mean ± SD of phospho-Rab10/total Rab10 and phospho-Rab12/total Rab12 ratios, which were quantified using the Image Studio software. Values were normalized to the average of Litter 1 wildtype MEFs treated with DMSO. Similar results were obtained in three independent experiments. (B–D) 6-month-old, littermate-matched wildtype (WT) and Rab29 knock-out (-/-) mice were administered with vehicle (40% (w/v) (2-hydroxypropyl)-ß-cyclodextrin) or 30 mg/kg MLi-2 dissolved in vehicle by subcutaneous injection 2 h prior to tissue collection. Forty micrograms of tissue extracts were analyzed by quantitative immunoblot as described in (A). Each lane represents tissue extract derived from a different mouse. Quantified data are presented as mean ± SD and values were normalized to the average of the wildtype, vehicle-treated mice. Data were analyzed using two-tailed unpaired t-test and there was no statistical significance between WT and Rab29 knock-out mice. The resulting P-values from the unpaired t-tests are (B) lungs P = 0.6585 (C) spleen P = 0.9318 (D) kidneys P = 0.4793.

Fig 5: Overexpression of Rab29 does not impact LRRK2-mediated Rab10 phosphorylation.(A–F) 6-month-old littermate-matched wildtype (WT) and homozygous, transgenic Rab29 overexpressing (Tg Rab29 Hom) mice were administered with vehicle (40% (w/v) (2-hydroxypropyl)-ß-cyclodextrin) or 30 mg/kg MLi-2 dissolved in the vehicle by subcutaneous injection 2 h prior to tissue collection. Forty micrograms of tissue extracts were subjected to quantitative immunoblot analysis with the indicated antibodies. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Quantified data are presented as the ratios of phospho-Rab10/total Rab10 and phospho-Rab12/total Rab12, calculated using the Image Studio software. Values were normalized to the average of the wildtype, vehicle-treated mice. Each lane represents a tissue sample from a different animal. Quantifications are presented as mean ± SD and data were analyzed using two-tailed unpaired t-test. There was no statistical significance in Rab10 phosphorylation between wildtype and homozygous, transgenic Rab29 overexpressing mice. The resulting P-values from the aforementioned statistical analyses of Rab10 phosphorylation between genotypes are as follows: (A) brain P = 0.0625 (B) large intestine P = 0.8026 (C) kidneys P = 0.7243 (D) spinal cord P = 0.3120 (E) spleen P = 0.7257 (F) lungs P = 0.1676. The resulting P-values from the statistical analyses of Rab12 phosphorylation between genotypes are: (A) brain P = 0.8545 (B) large intestine P = 0.1831 (C) kidneys *P = 0.0316 (D) spinal cord P = 0.3361.