FIG. 1.

Novel PKCs are involved in NF-κB and JNK activation in response to PMA in primary murine splenic B and T cells. (A) Splenocytes, MACS-separated Thy1.2-positive cells (Thy1.2+), or MACS-separated B220-positive cells (B220+) from either wild-type (BALB/c) or nude mice were analyzed for Thy1.2 and B220 expression by flow cytometry. (B) Whole cell extracts of B220- or Thy1.2-positive cells were analyzed for the expression of different PKC isoenzymes, as indicated. A nonspecific band that migrates slightly faster than PKCθ is indicated by a circle. (C and D) T cells from BALB/c mice and B cells from nude mice were pretreated for 30 min with the indicated concentrations of PKC inhibitors Gö6976 or rottlerin and subsequently stimulated with PMA. NF-κB activity was determined by EMSA (C) and IκBα degradation and JNK phosphorylation were determined by Western blotting (D). (E) Specificities of Gö6976 and rottlerin were confirmed in an in vitro kinase reaction using purified rat brain PKCs and myelin basic protein as a substrate.