(A) Immunofluorescence detection of endogenous SMAP1 in wild-type and Smap1–/– MEFs using an anti-SMAP1 antibody (green). Blue indicates DAPI staining. (B and C) Internalization of transferrin in wild-type and Smap1–/– MEFs. Cells were incubated with fluorescein-transferrin for the indicated times, and then surface-remaining transferrin was stripped off. The cells were then processed for analyses by (B) fluorescence microscopy or (C) flow cytometry. In C, the intensities of intracytoplasmic fluorescence were measured and expressed in relative arbitrary units. Independent cultures were prepared in triplicate from the indicated MEF clones, and averages ± SD are shown (n = 3). “No. 1,” “No. 8,” “No. 5,” and “No. 7” refer to the MEF cell line numbers. (D) Effects of siRNA against SMAP2. Wild-type MEFs were treated with or without siRNA against SMAP2 (2 differentially designed siRNAs, siRNA1 and siRNA2, were used). Protein lysates were prepared and processed for immunoblot analyses using anti-SMAP2 and anti-SMAP1 antibodies. (E) Effects of SMAP2 knockdown on transferrin incorporation. The Smap1+/+ and Smap1–/– MEFs were incubated with siRNA2 against SMAP2 and then with fluorescent transferrin and processed for immunofluorescence detection using an anti-SMAP2 antibody. The arrows indicate the reduction in fluorescence intensity from SMAP2, whereas DAPI staining indicates the location of cell nuclei. Scale bar: 10 μm.