Fig. 5.

The F56C mutation reduces mature PR release from a model precursor. a, b Schematic drawing showing miniprecursor distal and proximal cleavage sites and the F56 mutants tested. c Autoprocessing of L-MBP fusion precursors carrying the indicated mutations. Post-nuclear total lysates were resolved on SDS-PAGE and analyzed by Western blotting. Each blot was divided into two parts with 25 kD as the cutoff size and then probed separately with mouse Flag and GAPDH (top) or HA (bottom) antibodies. Both blots were probed with IR800 goat anti-mouse secondary antibody and scanned using a LI-COR Odyssey scanner. The brackets on the right indicate the autoprocessing products resulted from the proximal (P) and distal (D) site cleavages. The images were representative of seven experiments. [Note that the parental NL4-3 employed here encodes SFSF; the parental NL4-3 employed in Fig. 3 encodes SFNF, reflecting natural polymorphisms at residue 55]