Skip to main content
. 2014 Mar 7;289(16):11431–11442. doi: 10.1074/jbc.M113.524082

FIGURE 4.

FIGURE 4.

In vivo phosphorylation of Env7-HA expressed from a constitutive plasmid promoter is dependent on N-terminal cysteines. A, P13 membranous fraction of designated samples were incubated with or without an ATP regeneration system (left), in the presence or absence of hydroxylamine (HDX) (middle), in presence or absence of alkaline phosphatase (right), or in the presence or absence of phosphatase inhibitors and alkaline phosphatase (far right). Samples were resolved by low percentage SDS-PAGE and analyzed for mobility shift by Western blotting using anti-HA antibody. B, S0.4 fractions of the indicated samples were incubated in the presence or absence of an ATP regeneration system and analyzed as described in A. Hexokinase I (HKI) was used as a loading control (bottom). Bands of lower exposure blots were quantified as described under “Experimental Procedures,” and the extent of phosphorylation (upshifted species) was expressed as a percentage of the total (upshifted + non-upshifted) for each protein. Env7C14S/C15S-HA was stabilized with proteasomal inhibitor MG-132.