Figure 8

The association of NPM1 with RNA is attenuated in a NPM1c-positive cell line. (A) RNA was extracted from HL60 (expressing NPM1 (WT)) and OCI-AML3 (expressing NPM1 (WT) and NPM1c) cells. The expression of NPM1 (WT) and NPM1c was detected by RT-PCR. (B) Distribution of NPM1 in HL60 and OCI-AML3 cells prepared as in Fig. 2A. Then, NPM1 (WT) and NPM1c were detected by Western blotting. (C) HL60 and OCI-AML3 cells were lysed with buffer containing Triton X-100, SDS, and DOC. The lysate was immunoprecipitated with antibody against NPM1. NPM1 (WT) and NPM1c were detected by Western blotting with the indicated antibodies. (D,E) Triton X-100 soluble and insoluble lysates prepared from HL60 (D) and OCI-AML3 (E) cells were subjected to SEC. NPM1 (WT) and NPM1c were detected by Western blotting. RNA was extracted from each fraction and detected by ethidium bromide staining. (F) Interaction of NPM1 complex with RNA. The Triton X-100 soluble lysates from HL60 and OCI-AML cells were immunoprecipitated with anti-NPM antibody. NPM1 and extracted RNA were detected by immunoblot and ethidium bromide staining, respectively. (G) Quantification of the data from panel F. The normalized RNA levels coprecipitated with NPM1 are shown. The data represent the average of three independent experiments ± SEM for each condition.