Effect of Ras on PKC expression and translocation. (A) Representative Western blot data from day 7 cultures, fractionated into cytosolic and membrane components, and probed with the indicated antibody; c/nPKC P-Ser660 binds all conventional and novel isoforms of PKC only when phosphorylated at Ser660 (or equivalent residue). A total of 5 × 10⁴ cell equivalents were loaded in each lane. Note: PDK1 is a multiply phosphorylated protein that gives rise to species of different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); treatment of extracts with phosphatase gives rise to a single fast-migrating band (not shown). (B) Corresponding analysis at day 3 of culture (day 0 following infection). In each case, quantitation was carried out by densitometric analysis of band volumes (from at least 4 replicate experiments); *P ≤ .05; **P ≤ .01. Note that the values are normalized relative expression levels (compared with the equivalent control fraction). These are mean values and therefore may not exactly match the fold-changes shown in the corresponding representative data. To clearly show membrane-translocated species, some cytoplasmic banding is overexposed.