Figure 2. In vitro ERK phosphorylation by MEK in the presence of PEG-6000 used as a crowder agent.

(A) ERK was phosphorylated by MEK at the indicated concentration of PEG-6000 (v/v). The samples were subjected to Western blotting analysis with Phos-tag. (B) The fractions of phospho-isoforms of ERK determined in (A) are plotted against time. Lines represent the fractions of phospho-isoforms of ERK obtained by fitting the experimental data with the quasi-processive model in Fig. 1B. Raw and fitted data correspond to Exp #2 in Supplementary Dataset. (C) The crowding factor (c, green) and processivity (p, red) at each PEG-6000 concentration were determined by fitting as described in the text. Additional data are included in Supplementary Figure S1. Dashed lines are drawn for emphasis.