Expression of inactive mutant HRI increases the α and β globin contents of differentiating MEL cells.
(A) Expression of retrovirally transduced HRI in MEL cells. MEL cells expressing vector alone, K199R HRI, or Δ10 HRI were plated at 1 × 107 cells per 100-mm plate the night before treatment with 2% DMSO as indicated. Cells were counted and harvested on day 5. Cells were lysed at 1 × 108 cells/mL. Cytoplasmic extracts, equivalent to 2 × 106 cells, were used for anti-HRI Western blot analysis. Lanes 1 and 2 are uninfected MEL cell controls; lanes 3 and 4 are K199R-infected cell extracts; lanes 5 and 6 are Δ10 HRI-infected cell extracts; lane 7, vector-infected cell extracts. (B) α and β globin contents of MEL cells expressing mutant HRI. Lanes 2, 4, and 6 are induced with DMSO. Cytoplasmic extracts, equivalent to 5 × 105 cells, were separated by 15% SDS-PAGE and stained with Coomassie blue. The positions of α and β globin were marked. Lane 1, rabbit reticulocyte lysates. Lanes 2 and 3, cell extracts; lanes 4 and 5, K199R HRI-expressing cell extracts; lanes 6 and 7, Δ10 HRI-expressing cell extracts; lanes 3, 5, and 7 are treated with DMSO.
