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. 2022 Aug 25;144(37):16930–16952. doi: 10.1021/jacs.2c05499

Figure 8.

Figure 8

XL01126 degrades LRRK2 in human peripheral blood mononuclear cells (PBMCs) derived from healthy donors and mouse bone marrow-derived macrophages (BMDMs). Representative Western blotting of total LRRK2, LRRK2-pSer935, pRab10, Rab10 total, and GAPDH levels following treating the PBMCs with XL01126 and cis-XL01126 at the indicated concentrations for 4 h (A) and 24 h (B). The relative LRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/GAPDH or Rab10-pThr73/total Rab10, respectively, and the ratios were normalized to the DMSO-treated samples. The relative LRRK2 and pRab10 protein levels were plotted against the compound concentration and fitted against “nonlinear regression, one site-fit log IC50” in GraphPad to obtain the DC50 and EC50 values. Data points are presented as mean ± standard error of the mean (SEM) from three biological independent replicates. (C) Representative Western blotting of total LRRK2, LRRK2-pSer935, pRab10, Rab10 total, and GAPDH levels following treating the PBMCs with 300 nM of XL01126 and cis-XL01126 for the indicated time periods. The relative LRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/ GAPDH or Rab10-pThr73/total Rab10, respectively, and the ratios were normalized to the DMSO-treated samples. The relative LRRK2 and pRab10 protein levels were plotted against the treatment time and were fitted against “nonlinear regression, one-phase decay” in GraphPad to obtain the half-life (T1/2) values. Data points are presented as mean ± SEM from three biological independent replicates. (D) Representative Western blotting of LRRK2 total and tubulin levels after treating BMDMs with XL01126 and cis-XL01126 for 4 h. The relative LRRK2 levels were obtained by quantifying the ratios of total LRRK2/tubulin and normalized to the DMSO-treated samples. The relative LRRK2 levels were plotted against the compound concentration and fitted against “nonlinear regression, one site-fit log IC50” in GraphPad to obtain the DC50 values.