FLT3 receptor membrane expression, tyrosine phosphorylation, and inhibition of receptor tyrosine phosphorylation by PKC412 in 32D cells transfected with FLT3-ITD and FLT3-ITD-N676K. Murine 32D cells expressing FLT3-ITD and FLT3-ITD-N676K were generated as described in “Materials and methods.” (A) Membrane expression of FLT3 was determined by flow cytometry using a specific anti-FLT3 antibody. FLT3 receptors were expressed at comparable levels in 32D-ITD and 32D-ITD-N676K cells (B). FLT3 tyrosine phosphorylation was analyzed by Western blotting using phosphotyrosine-specific FLT3 antibodies. FLT3 tyrosine phosphorylation levels in 32D-FLT3-ITD and 32D-FLT3-ITD-N676K cells did not reveal apparent differences. (C) Inhibition of FLT3 tyrosine phosphorylation by PKC412 was analyzed in 32D cells expressing FLT3-ITD or FLT3-ITD-676 mutant receptors. Cells were treated with or without PKC412 at various concentrations for 30 minutes, and FLT3 tyrosine phosphorylation was analyzed by immunoblotting using phosphotyrosine-specific FLT3 antibodies. To control equal loading, the blot was stripped and reprobed using anti-FLT3 and antiactin antibodies, respectively. On treatment with 10 nM PKC412, densitometric analysis revealed more than 80% inhibition of tyrosine-phosphorylated FLT3 levels in 32D-FLT3-ITD cells, whereas in 32D-FLT3-ITD-N676K cells inhibition was 19%.