Expression of NPMc⁺ and NPM in hMRP8-NPMc⁺ and hMRP8-NPM transgenic mice. (A) Schematics of hMRP8-NPMc⁺ and hMRP8-NPM constructs used to generate NPM transgenic lines. (B) Southern blot analysis using the 3′ probe of BamHI-digested tail DNA from hMRP8-NPMc⁺ founders. Positive founders were confirmed by PCR as illustrated below the Southern blot panel. (C) Semiquantitative RT-PCR analysis of NPMc⁺ and NPM mRNA levels from whole BM cells of different founders. Red arrows represent the transgenic lines used for detailed analysis. NT indicates nontransgenic control. (D) Semiquantitative RT-PCR analysis of NPMc⁺ mRNA levels from sorted populations. Myeloid: Mac-1⁺Gr-1⁺ BM cells; B-cell: B220⁺CD19⁺ splenocytes; T-cell: CD4⁺ splenocytes. (E) Western blot of NPMc⁺ and NPM expression in whole BM cells of NPMc⁺ 28 (left panels) and NPM 65 (right panels) animals. A monoclonal Flag antibody (top left) and a polyclonal NPMc⁺-specific antibody (bottom left) were used to detect NPMc⁺ levels. *Nonspecific band. A polyclonal Flag antibody (top right) and a monoclonal NPM antibody were used to detect both overexpressed human NPM and endogenous mouse Npm levels. C indicates control lysates from HEK293 cells transfected with an NPMc⁺ expression vector; NPM 69, a negative control (an NPM transgenic mouse that was not found to overexpress Npm at the level of RNA or protein). (F) Western blot analysis of NPMc⁺ and NPM expression on purified nuclear and cytoplasmic fractions of Mac-1⁺Gr-1⁺ sorted granulocytes from BM of NT, NPM 65, and NPMc⁺ 28 animals. A polyclonal Flag antibody was used to specifically recognize the transgenic wild-type NPM or NPMc⁺ mutant. A monoclonal NPM antibody was used to detect the endogenous mouse Npm levels. Anti-LaminB1 and anti-Hsp90 assess the purity of the nuclear (N) versus the cytoplasmic (C) fraction, respectively.