American Society of Hematology

Fig. 2.

$Fig. 2. Cleavage of eALAS by a proteolytic activity in bone marrow erythroid cells. / (A) Blood (1 mL) was separated on a 3-mL Ficoll-Hypaque gradient. The high-density fraction was resuspended in medium and divided into 2 tubes to which 7.4 MBq (200 μCi)35S-methionine was added. A 300-μL nonlabeled bone marrow lysate (1.5 × 108 cells) (lane 2) or medium alone (lane 1) was added. Both samples were incubated at 37°C for 3 hours, and eALAS proteins were subsequently immunoprecipitated and gel-separated. (B) Erythroid and granulocytic cells were isolated from the bone marrow by mAbs coupled to magnetic beads (“Materials and methods”). We incubated 80-μg lysates from these fractions with a labeled blood lysate for 30 minutes at 37°C, and eALAS proteins were subsequently immunoprecipitated and gel-separated. The far-right lane represents a lysate after depletion of erythroid cells.$

Cleavage of eALAS by a proteolytic activity in bone marrow erythroid cells.

(A) Blood (1 mL) was separated on a 3-mL Ficoll-Hypaque gradient. The high-density fraction was resuspended in medium and divided into 2 tubes to which 7.4 MBq (200 μCi)³⁵S-methionine was added. A 300-μL nonlabeled bone marrow lysate (1.5 × 10⁸ cells) (lane 2) or medium alone (lane 1) was added. Both samples were incubated at 37°C for 3 hours, and eALAS proteins were subsequently immunoprecipitated and gel-separated. (B) Erythroid and granulocytic cells were isolated from the bone marrow by mAbs coupled to magnetic beads (“Materials and methods”). We incubated 80-μg lysates from these fractions with a labeled blood lysate for 30 minutes at 37°C, and eALAS proteins were subsequently immunoprecipitated and gel-separated. The far-right lane represents a lysate after depletion of erythroid cells.

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