Biotin labelling and pulldown of untagged IFITM1.

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.