d off with TBS and the cells lysed with 100 ml Triton X-100 lysis buffer. The whole cell lysate was added to a 25 ml NeutrAvidin agarose bead pellet and incubated for 3 h at 4uC on a rotor. Beads were then pelleted and the unbound material collected. The bead pellet was then washed with Triton X-100 lysis buffer, TBS and a TE buffer, prior to elution of proteins in 70 ml of 16 reducing LSB and heating at 95uC for 10 min. Eluate was collected and the elution procedure repeated. Equivalent volumes of whole cell lysate, unbound material and eluate from the NeutrAvidin beads were separated by 15% SDS-PAGE and western blotted as previously. Calreticulin was used as a loading control and detected with an anti-calreticulin antibody diluted 1:5000 in 1% BSA-TBST. cells. Furthermore, the C-terminal HA-tag is accessible on intact cells and therefore located on the extracellular surface of the plasma membrane. Co-staining cells with fluorescent wheat germ agglutinin further confirmed this plasma membrane localisation. No anti-HA labelling was seen on intact IFITM2-HA expressing cells, in keeping with the notion that this protein is located on intracellular membranes. A similar result PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19664521 was seen for IFITM3-HA, although a low number of cells did show some surface labelling with anti-HA antibodies. This suggested that although the majority of IFITM3-HA is located on intracellular membranes, in some cells IFITM3-HA is either mis-sorted and/or is expressed at the cell surface where the C-terminal HA epitope is accessible. IFITM N-terminal domains reside intracellularly The observation that the HA-tag of IFITM1 is accessible at the surface of intact cells prompted us to investigate the topology of the protein. We used two commercially available antibodies against the hu IFITM1 and IFITM3 NTDs. The IFITM1 NTD antibody was raised against a peptide encoding the first 35 amino acids of hu IFITM1. This domain has 69% and 75% amino acid identity with hu IFITM2 and IFITM3, respectively. The IFITM3 NTD antibody was raised against a peptide with a sequence corresponding to the first 30 amino acids of hu IFITM3. Hu IFITM1 has an Nterminal 21 amino acid truncation compared to IFITM3, 
so contains only 9 amino acids overlapping with this domain. However, there is 90% amino acid identity between the first 30 amino acids of hu IFITM3 and IFITM2. To determine the specificity of the NTD antibodies, both were tested on samples from the HA-tagged IFITM1-3 expressing A549 cells by western blot analysis. Anti-IFITM1-NTD detected PG 490 IFITM1-HA and IFITM3-HA, but not IFITM2-HA. Anti-IFITM3-NTD detected both IFITM3-HA and IFITM2-HA, but not IFITM1-HA. No IFITM protein was detected in control A549 cells with either antibody by western blot. Multiple bands in the range of 1217 kDa were seen with the NTD antibodies, but not the anti-HA, indicating possible post-translational modification of the proteins. When intact IFITM1, 2 or 3 cells were labelled with antiIFITM1-NTD antibody, no staining was seen, suggesting intracellular NTDs. Following detergent treatment, all three IFITM expressing lines were labelled, though a weaker signal was seen for IFITM2, as expected from the western blot. By immunofluorescence, the anti-IFITM3NTD antibody gave a low signal in the untransfected A549 control and IFITM1-HA cells, consistent with the low level of endogenous IFITM2 expression detected by qRT-PCR. However, the antibody clearly detected IFITM2-HA and IFITM3-HA. The observed cellular distributio
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