FIG. 3.

Cytosolic Grp75 is increased in the heavy MAM fraction of HCMV-infected LE-HFFs at 8, 24, and 96 hpi. LE-HFFs were infected with HCMV, harvested at 8 or 24 hpi (A) or 96 hpi (B), and fractionated as described for Fig. 1. Proteins (20 μg) in the microsomal, heavy MAM (6,300 × g), and mitochondrial fractions were separated by SDS-PAGE and probed by Western blots using anti-Grp75 antibody (1:1,000). (C) Sensitivity of Grp75 in heavy MAM to proteinase K digestion. Twenty-microgram amounts of the microsomal, heavy MAM, or mitochondrial fraction from LE-HFFs transiently expressing gpUL37 were untreated (−) or treated (+) with proteinase K (3 μg/20 min on ice). Following digestion and inactivation with 1 μM phenylmethylsulfonyl fluoride, the proteins were resolved by SDS-PAGE, blotted, and reacted with anti-Grp75 antibody as described for panels A and B. Grp75 is indicated by arrowheads.