Fig. 3. The effects of PKC inhibitors on PKC activation and limb development. (A) Western blot (top panel) and quantification (lower panel) of activated PKC/PKCßII in primary limb bud cell cultures treated with PKC inhibitors. 1, medium only; 2, 100 nM Go 6976; 3, 1 µM Go 6976; 4, 13 µM chelerythrine chloride; 5, 130 µM chelerythrine chloride; 6, 30 µM sphingosine; 7, 300 µM sphingosine. In all cases, except for the low dose treatment with chelerythrine chloride, PKC inhibitors were effective in reducing PKC/PKCßII autophosphorylation. Quantification of PKC/PKCßII autophosphorylation was achieved by densitometry. Percent activity was normalized to PKCß detected by a pan PKCß antibody. Lane 1 (no inhibitor added) was defined as 100% activity. (B) Treatment of the presumptive wing bud with chelerythrine chloride produced a completely truncated wing. The scapula (s) was shortened and the clavicle (cl) had a small protrusion (arrow). (C) Contralateral wings were always normal showing humerus (h), radius (r), ulna (u) and digits 2, 3, and 4.