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. 2004 May;24(9):3827–3837. doi: 10.1128/MCB.24.9.3827-3837.2004

FIG. 5.

FIG. 5.

HOXA9 is phosphorylated at S204 in vivo and this phosphorylation is mediated by PKC. (A) Cytoplasmic or nuclear extracts of PLB985 cells were immunoprecipitated with goat α-HOXA9 antibody (αHOXA9#1). Western blot analysis of the immunoprecipitates using affinity-purified chicken α-HOXA9 antibody (αHOXA9#2) showed expression of HOXA9 predominantly in the nuclear extract and to a lesser extent in the cytoplasmic extract. HOXA9 immunoprecipitates from cytoplasmic and nuclear extracts of TPA-stimulated PLB985 cells, treated with or without phosphatase, were probed with α-pS204-HOXA9 antibody (#2). (B) HOXA9 protein from cytoplasmic and nuclear extracts from U937 and PLB985 cells treated with TPA was precipitated with αHOXA9#1 and detected with a rabbit affinity-purified α-HOXA9 antibody (#3) or a different α-pS204-HOXA9 antibody (#1). (C) Immunoprecipitates from nuclear extracts of U937 cells contained HOXA9 protein that was immunoreactive with αHOXA9#2 antibody. HOXA9 immunoprecipitates, from nuclear extracts of TPA-stimulated PLB985 and U937 cells, treated with or without phosphatase, were probed with α-pS204-HOXA9 antibody. (D) PLB985 cells stimulated with either TPA or control were pretreated with or without the PKC inhibitor bisindolylmaleamide1 (5 μM). Cytoplasmic and nuclear extracts were immunoprecipitated with αHOXA9#1, and analyzed by Western blotting with either αHOXA9#2 or α-pS204-HOXA9. Change in HOXA9 phosphorylation level was calculated by NIH ImageQuant analysis.