The BH3-only protein Bmf accumulates in granulocytes in culture.

(A) Sorted granulocytes from the bone marrow of C57BL/6 mice were cultured in simple medium for 24, 48, or 72 hours. Survival was assessed by trypan blue exclusion in a hemocytometer. Cells were harvested, lysed, and proteins subjected to Western blot analysis using antibodies specific for the BH3-only proteins Bim, Bid, or Bmf. Analysis of Hsp70 expression was performed as a loading control. Filters were sequentially probed, stripped, and reprobed (first Bmf, then Bim, then Bid, then Hsp70 or Puma, then Noxa, then Bad, then Hsp70). Data show 1 representative of 3 independent experiments. (B) Sorted granulocytes from Bim-deficient andbcl-2 transgenic mice were cultured in simple medium, and Bmf protein expression was analyzed as in panel A. Data show 1 of 2 independent experiments.