However, we can facilitate a comparison between autophosphorylation and 4E-BP phosphorylation by using recombinant LRRK2 fragment of known concentration and by analyzing kinase reaction products on the same gel. Threefold higher incorporation of 33P from labeled ATP into LRRK2 itself rather than 4E-BP, even when the later is present at more than a hundred-fold molar excess, demonstrates that the LRRK2 autophosphorylation reaction is much more efficient under these conditions. One limitation of this experiment is that a truncated LRRK2 Doxifluridine version was used, as this construct is more soluble and active than full-length protein. However, in cell lines expressing full length LRRK2 that was active when isolated and tested in vitro for autokinase activity, we did not see any evidence of 4E-BP phosphorylation either. These results were also strengthened by measuring the stoichiometry of phosphorylation for LRRK2 and 4E-BP in the same reaction, where we found that LRRK2 Ceftizoxime sodium incorporated phosphate more efficiently than 4E-BP again despite the latter being present at molar excess. Two further results give pause to the conclusion that 4E-BP is an authentic substrate for LRRK2. First, MAPK14/p38a and STK3 are capable of phosphorylating 4E-BP in vitro. As we have not performed an analysis of the complete kinome, we cannot be certain how many kinases can phosphorylate T37/T46 of 4E-BP but because other kinases can trigger phosphorylation of this site, the measurement of it in cell or tissue lysates is not a simple measure of LRRK2 activity per se. Second, in HEK 293FT inducible cell lines and in transiently transfected HEK 293FT cells we did not find any significant 4E-BP phosphorylation changes by wild-type LRRK2 or two different pathogenic mutants G2019S and R1441C, while MAPK14/P38a was able to efficiently phosphorylate 4E-BP in these cells. A caveat to these assays is that we cannot prove that LRRK2 is active in these cells without a direct marker of LRRK2 phosphorylation, although we were able to extract LRKK2 and measure autophosphorylation activity ex vivo.