American Society of Hematology

Fig. 4.

$Fig. 4. Expression of Nramp2 variants in stably transfected CHO cells. / (Left panels) Mutants at conserved charged residues in TM domains; (right panels) multiple mutants at the conserved histidines in TM6. Crude membrane fractions (A) or total cell lysates (B) were prepared from various transfected cell clones (identified) and separated by SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed using a mouse monoclonal anti-cMyc antibody directed against a corresponding epitope tag inserted in-frame in the Nramp2constructs. Controls include cells transfected with the plasmid alone (pCB6) and cells expressing low (N2GG) or high amounts (N2.3) of wild-type Nramp2.13 Molecular mass markers are identified in kilodaltons to the left of the immunoblots. (B) Prior to electrophoresis, intact cells were labeled with biotin and disrupted with lysis buffer (upper panels). Labeled cell surface proteins were then isolated with strepavidin beads (lower panels).$

Expression of Nramp2 variants in stably transfected CHO cells.

(Left panels) Mutants at conserved charged residues in TM domains; (right panels) multiple mutants at the conserved histidines in TM6. Crude membrane fractions (A) or total cell lysates (B) were prepared from various transfected cell clones (identified) and separated by SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed using a mouse monoclonal anti-cMyc antibody directed against a corresponding epitope tag inserted in-frame in the Nramp2constructs. Controls include cells transfected with the plasmid alone (pCB6) and cells expressing low (N2GG) or high amounts (N2.3) of wild-type Nramp2.13 Molecular mass markers are identified in kilodaltons to the left of the immunoblots. (B) Prior to electrophoresis, intact cells were labeled with biotin and disrupted with lysis buffer (upper panels). Labeled cell surface proteins were then isolated with strepavidin beads (lower panels).

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