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. Author manuscript; available in PMC: 2014 Nov 13.
Published in final edited form as: Cell Host Microbe. 2013 Nov 13;14(5):510–521. doi: 10.1016/j.chom.2013.10.011

Figure 4. HA transport to the cell surface is Vps4-independent but Tsg101-dependent.

Figure 4

(A) Immunodepletions from MDCK cytosol performed with anti-Vps4 or anti-Tsg101 antibodies on Protein A beads. Beads without antibodies were used as control. Equivalent fractions of input and flow-through were probed for Vps4 and Tsg101 respectively. GAPDH was used as a loading control to verify that depletion was specific. (B) 1 × 106 MDCK cells were infected with Influenza A/WSN/33 at an MOI of ~0.5 for 5 hours and radiolabeled with [35S]-cys/met. Cells were permeabilized with 100 nM PFO and supplemented with (i) concentrated MDCK cytosol, (ii) MDCK cytosol treated with control beads, (iii) cytosol depleted of Vps4 (iv) cytosol supplemented with GTPγS and chased for 30 and 60 mins. Control cytosol (lanes 1–3), cytosol containing Vps4 (lanes 4–6) and cytosol lacking Vps4 (lanes 7–9). Cytosol supplemented with GTPγS (lanes 10–12). (C) 1 × 106 MDCK cells were infected with Influenza A/WSN/33 at an MOI of ~0.5 for 5 hours and radiolabelled with [35S]-cys/met. Permeabilized cells were supplemented with (i) concentrated MDCK cytosol either mock depleted or Tsg101-deficient (ii) MDCK cytosol treated with IFN-I (iii) MDCK cytosol treated with IFN-I mixed with Ubp43 and either mock depleted or Tsg101-deficient (iv) cytosol supplemented with GTPγS and chased for 30 and 60 mins. Untreated cytosol containing Tsg101 (lanes 1–3); cytosol lacking Tsg101 (lanes 4–6). Cytosol from IFN treated cells treated with Ubp43, containing Tsg101 (lanes 10–12) or Tsg101 deficient cytosol (lanes 13–15). Cytosol supplemented with GTPγS (lanes 16–18). Autoradiograms are representative of atleast 3 independent experiments. (D) HEK293T cells were co-transfected with HA-ISG15 and either empty vector or Ubp43 de-ISGylase. Transfected cells were either untreated or treated with type-I IFN for 6 hours (100 U/ml) and lysed in NP-40. Lysates were immunoblotted with anti-HA antibodies. ISGylated products were immunoprecipitated with anti-HA and probed with anti-Tsg101.