p62, phospho-PKCζ/PKCζ, and VCAM-1 expression in MM stromal cells. Stromal cells (1°) from 13 patients and 11 healthy controls were cultured in 12-well plates for 3 days, and an aliquot of each sample was serum-starved for 12 hours and then exposed to serum for 10 minutes at the end of culture period. Levels of pPKC-ζ were determined in serum-starved samples, while PKCζ, actin, and p62 were determined in the matched unstarved samples by Western blot analysis as described in “Methods.” Equal amounts of protein were loaded in all lanes. (A) Typical results for PKCζ phosphorylation and p62 for 4 MM patients and 2 healthy subjects are shown. The relative levels were compared with healthy patient 2, who expressed the lowest level of p62 and pPKCζ. The black marks reflect splicing of the results to group normal and myeloma subjects together, specifically normal 2 was moved next to normal 1 for the pPKCζ blot. (B) The ratio of p62 and phospho-PKCζ/PKCζ in MM compared with normal stromal cells for 13 MM patients and 11 healthy subjects. Results represent the mean (± SEM). *P < .01.