To further compare the degradation profiles of XL01134 and XL01126 with that of SD75, a time-dependent degradation assay was performed in MEFs using Western blotting (Figure ). SD75 was shown to degrade WT LRRK2 and G2019S LRRK2 at 1 µM in a time-dependent manner with moderate Dmax (52% for WT LRRK2 and 81% for G2019S LRRK2) and half-lifes (T1/2) against WT LRRK2 (5.1h) and G2019S LRRK2 (1.4h). In contrast, XL01134 and XL01126 degraded LRRK2 at higher rates, achieved higher Dmax values at only 300 nM, a concentration at which SD75 barely degraded LRRK2. Remarkably, XL01126 presented an improved profile (Dmax, WT = 82%, Dmax, G2019S = 92%, T1/2, WT = 1.2h, T1/2, G2019S = 0.6h) when compared to XL01134 (Dmax, WT= 75%, Dmax, G2019S = 82%, T1/2, WT= 2.7h, T1/2, G2019S = 1.4h). With the shortest degradation half-lives and highest degradation percentage, XL01126 emerged as the most efficient and fastest degrader among the three. The timedependent pRab10 dephosphorylation correlates well with the LRRK2 degradation (Figure , 6B and 6C). XL01126 dephosphorylated pRab10 the fastest with T1/2, pRab10 at 0.7h and 0.3h in WT and G2019S LRRK2 MEFs, respectively. This was followed by XL01134, which induced 50% reduction in Rab10 phosphorylation after 2.1h and 0.3h in WT and G2019S LRRK2 MEFs, respectively. In contrast, SD75 exhibited the slowest inhibition of pRab10 (T1/2, pRab10 = 6.7h on WT MEFs and 1.1h on G2019S LRRK2 MEFs). Figure . Time-dependent LRRK2 degradation, LRRK2 dephosphorylation and pRab10 dephosphorylation by SD75, XL01134, and XL01126. Representative Western blots of total LRRK2, LRRK2-pSer935, Rab10-pThr73, Rab10 total, and Tubulin levels after treating the WT and G2019S LRRK2 MEFs with SD75 (A), XL01134 (B), or XL01126 (C) at the indicated concentrations for the indicated period of time. The relative LRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/Tubulin 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 "non-linear regression, one phase decay" in GraphPad to obtain the half-life (T1/2) values. Data were obtained from two independent biological experiments. XL01126 surpassed its warhead and negative PROTAC cis-XL01126 in inhibiting downstream signaling in G2019S LRRK2 MEFs. Representative Western blots of total LRRK2, LRRK2-pSer935, pRab10, Rab10 total, and Tubulin levels following the treatment of G2019S LRRK2 MEFs with HG-10-102-01 (A), XL01126 (A and B), and cis-XL01126 (B) at the indicated concentrations for 4h. The relative LRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/Tubulin 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 compounds concentration and fitted against "non-linear regression, one site-fit LogIC50" in GraphPad to obtain the DC50 and EC50 values. Data were obtained from two independent biological experiments.