Trypsin cleavage of HA-tagged IFITM1.

<p>A) Predicted trypsin cleavage sites in hu IFITM1 CTD (Model 3, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104341#pone-0104341-g001" target="_blank">Fig. 1</a>). B) IFITM1-HA cells were treated with exogenous trypsin for 5 to 30 mins at 37°C. The trypsin was inactivated with soybean trypsin inhibitor, the cells were lysed and the cellular proteins separated by SDS-PAGE. After transfer, proteins were identified with anti-IFITM1-NTD (i) and anti-HA (ii) antibodies. VDAC was used as a loading control (iii). Control samples were untreated (UN), or treated with SBTI-inactivated trypsin (IN). In the overlay image, red represents anti-IFITM1-NTD labelling and green represents anti-HA labelling (iv).</p

Cellular distribution of the human IFITM proteins.

<p>C-terminal domain HA-tagged IFITM1, IFITM2, IFITM3 and control A549 cell lines were stained intact, or following permeabilisation, with an anti-HA antibody and a secondary anti-rat Alexa-488 antibody. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA have distinct cellular distributions in permeabilised cells. E) Intact A549 cells (no detergent treatment) show no specific staining. F) Intact IFITM1-HA cells show positive cell surface HA staining. G) Intact IFITM2-HA cells show no detectable HA staining. H) Although the majority of intact IFITM3-HA cells show no anti-HA labelling, a minority (<1%) show low-level positive staining. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells using a confocal microscope. All images were taken using the same microscope settings and the levels adjusted uniformly. Scale bar represents 15 µm.</p

Image analysis of anti-IFITM3-NTD antibody and anti-HA antibody co-labelling.

<p>Relative areas of each colour were calculated as described in <i>materials and methods</i>. Red represents anti-IFITM3-NTD labelling, green represents anti-HA labelling and yellow represents overlap of the two stains. Error given is of the standard deviation.</p

Co-staining with N- and C-terminal antibodies.

<p>Permeabilised IFITM2-HA (A) and IFITM3-HA (B) expressing cells were stained with antibodies against the C-terminal HA-tag (green [Alexa-488]) and the NTD, using the anti-IFITM3-NTD antibody (red [Alexa-647]). Images are single optical sections (0.25 µm thick) through the cells. Scale bars represent 15 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104341#pone-0104341-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104341#pone-0104341-t003" target="_blank">3</a> for image analysis.</p

Antibody feeding of IFITM expressing cells.

<p>Live A549-IFITM-HA cells were incubated with 5 µg/ml anti-HA at 37°C to allow endocytosis of bound antibody molecules. Subsequently, the cells were washed, fixed, permeabilised and incubated with an anti-rat Alexa-488 conjugate. A) Control A549 cells show no specific staining. B) In IFITM1-HA cells the majority of labelling is at the plasma membrane. C) IFITM3-HA cells that were not incubated with anti-HA antibody show no labelling. For IFITM2-HA and IFITM3-HA expressing cells (D and E) the majority of cells are not labelled, however in both cases a minority of cells do show punctate labelling indicative of IFITM-mediated internalisation of anti-HA antibodies. In D and E, the boxed region has been enlarged. All images were taken using the same microscope settings and adjusted uniformly. Scale bars represent 15 µm.</p

Analysis of IFITM NTD antibodies.

<p>Two commercially available antibodies targeting either the IFITM1-NTD or the IFITM3-NTD were screened by western blot to assess specificity using HA-tagged IFITM1-3 (M1, M2 and M3) cell lines along with control A549 cells. Proteins were also identified using the HA epitope. Blots were imaged on a Li-COR Odyssey system that uses far-red fluorophore conjugated secondary antibodies. In the overlay image, red represents anti-IFITM-NTD labelling and green represents anti-HA labelling. A) Anti-IFITM1-NTD detects IFITM1 and shows cross-reactivity with IFITM3. B) Anti-IFITM3-NTD detects IFITM3 and has cross-reactivity with IFITM2. VDAC was used as a loading control.</p

IFITM membrane topology models.

<p>In Model 1, the N- and C-terminal domains are extracellular and are connected by two transmembrane domains (M1 and M2) and the conserved intracellular loop (CIL). In Model 2 the two hydrophobic domains (M1, M2) do not span the membrane, resulting in NTD, CTD and CIL domain being located in the cytoplasm. In Model 3, the NTD and CIL domain are intracellular, suggesting M1 does not span the membrane, but the CTD is located on the extracellular side of the membrane and requires that M2 spans the membrane.</p

Biotin labelling and pulldown of untagged IFITM1.

<p>Untagged, wild type IFITM1 or IFITM1-Vstop expression plasmids were transfected into HEK293T cells. After two days the cells were labelled with cell impermeable Sulfo-NHS-SS-Biotin prior to incubation with NeutrAvidin agarose beads. A) Diagram to show the exposed CTD of IFITM1, with the targeted K122, or IFITM1-Vstop. B) Western blots probed with anti-IFITM1-NTD; WCL – whole cell lysates, UB – material that remained unbound by NeutrAvidin, PD1 and PD2 – two rounds of elution of protein from the NeutrAvidin beads. Gel i shows samples from cells labelled with biotin, gel ii shows unlabelled samples and gel iii shows samples from mock transfected HEK293T cells that were treated with Sulfo-NHS-SS-Biotin. NB. The elution step detached some NeutrAvidin monomers from the beads. These run at approximately 14 KDa and are seen as background bands in the western blots (labelled ‘NeutrAvidin’). Calreticulin was used as a loading control and negative control for pulldown specificity. C) Western blot comparing the wild type IFITM1 (M1) with IFITM1-Vstop (Vstop), along with mock transfected HEK293T cells. D) Western blots probed with anti-IFITM1-NTD for whole cell lysates (i) material that remained unbound to NeutrAvidin (ii) and protein eluted from the NeutrAvidin beads (iii). As previously, NeutrAvidin monomers were eluted, and have the same molecular weight at IFITM1-Vstop. This can be clearly seen in the pulldown blot due to the presence of a band in the unlabelled lane.</p

Immuno-gold labelling of A549 IFITM1-HA cell cryo-sections.

<p>Cryo-sections of the IFITM1-HA cells were labelled with anti-HA antibodies and Protein A gold. A) Plasma membrane labelling. B and C) Plasma membrane and multi-vesicular body labelling. D) Plasma membrane and Golgi apparatus labelling. Scale bars represent 200 nm.</p

qRT-PCR primers.

<p>A list of the primers used for qRT-PCR. F′ and R′ stand for forward and reverse, respectively.</p