Western blot analysis of Nramp2/DMT1 association with latex particle–containing phagosomes formed in Sertoli cells and macrophages.

(A) Latex bead–containing phagosomes were purified from cell homogenates of TM4 Sertoli cells and from RAW macrophages. Crude membrane extracts (m) from control CHO (CHO LR73), TM4, and RAW cells or from stable c-Myc–Nramp1 or c-Myc–Nramp2 CHO or RAW transfectants22 or from latex bead–containing phagosomes (p) isolated from either TM4 (pTM4) or from RAW cells (pRAW) were separated by SDS-PAGE. Immunoblots were sequentially analyzed with a rabbit antimouse Nramp2/DMT1 antiserum (upper panel), a rat anti-Lamp1 (middle panel), and a mouse anti–c-Myc (lower panel) monoclonal antibody and revealed by ECL. (B) Crude membrane proteins (m) from control CHO and WEHI cells, c-Myc–Nramp2 CHO transfectants, murine Sertoli cell line 15-P1, or latex bead phagosomes isolated from 15P-1 (p15P-1) were analyzed by immunoblotting with a rabbit anti-Nramp2/DMT1 antiserum. (C) Latex bead phagosomes (p) from TM4 cells, together with membrane fractions (m) from control TM4, and CHO cells and from c-Myc–Nramp2 CHO transfectants were analyzed by immunoblotting with antibodies directed against Nramp2/DMT1, Lamp1, and the transferrin receptor (TfR). (D) Crude membrane proteins (m) from CHO or WEHI cells (used as negative controls) or from c-Myc–Nramp2 CHO transfectants (used as positive controls) or from latex bead–containing phagosomes isolated from RAW (pRAW) of TM4 cells (pTM4) were analyzed with an anti-EEA1 antibody.