Anti vps35

Antibodies and reagents used were anti-JIP4 (Cell Signaling, cat #5519, western blotting 1:1000, immunofluorescence 1:100 or 1:500; Fig. 2C). Anti-Phafin2 (Sigma-Aldrich HPA024829; 1:1000 western blotting); anti-GFP (Roche, cat #11814460001, western blotting 1:1000); anti-EEA1 antiserum (gift from Ban-Hock Toh of Monash University, Australia; immunofluorescence 1:160,000); anti-VPS35 (Abcam cat #ab10099, immunofluorescence 1:200) and phalloidin CF568 (Biotium Cat #00044, 1:200).

3.5 × 10⁵ clonal HeLa-tTA cells expressing pT-JX2 or pT-FA in the absence of doxycycline were seeded in 6 cm² plates and infected with HPV16 PsV at MOI of 150. For knockdown experiments, 2 × 10⁵ HeLa S3 cells expressing JX2 or empty vector were plated in 6 cm² plates, and after 16 h cells were transfected with 10 nM siRNA. Forty-eight h later, cells were infected with HPV16 PsV at MOI of 150. At 12 h.p.i., cells were washed with cold PBS and lysed with 400 μL ice-cold lysis buffer (20 mM HEPES [pH 7.4], 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT and 0.2% Triton X-100) supplemented with 1X Halt protease and phosphatase inhibitor cocktail (Thermo Fisher). The lysate was clarified by centrifugation at 14,000 rpm for 20 min at 4°C. Immunoprecipitation was carried out by adding anti-FLAG affinity gel (Sigma #A2220) or anti-VPS35 (Abcam, ab10099) antibody and protein A/G-PLUS-agarose beads to the clarified cell lysates at 4°C for 4 h, followed by 5 washes with lysis buffer. Protein complexes were eluted from the beads by heating to 100°C in 2X SDS sample buffer. Eluates and the Input were normalized for total extracted protein, as determined by bicinchoninic acid (BCA) protein assay and analyzed by SDS-PAGE followed by immunoblotting or staining with Silver Stain Kit (Pierce).