WJ460 (DMSO) was purchased from Glixx Laboratories Inc. or MedChem Express; A-485 (DMSO), vacuolin-1 (DMSO) and apilimod (DMSO) were purchased from MedChem Express; EGA (DMSO), leupeptin (H2O) and pepstatin A (DMSO) were purchased from MilliporeSigma; E64D (DMSO) was purchased from Cayman Chemical. Compounds were purchased in solution or reconstituted and were aliquoted and stored at -20°C or -80°C as recommended by the manufacturers. Viability following treatment with WJ460 was assessed using alamarBlue (Bio-Rad) according to the manufacturer’s instructions. Cells were transfected using Turbofect (Thermo Fisher Scientific) according to the manufacturer’s instructions and harvested 24 h later. Whole cell extracts were prepared, and western blotting was done as described [98 (link)]. Affinity enrichment of HBZ using GST-KIX was done as described [34 (link)]. Antibodies used were as follows: anti-HTLV-1 Env (ARP-1578) and anti-Tax (hybridoma 168B17-46-92) were obtained from NIH AIDS Research and Reagent Program; anti-HBZ has been described [99 (link)]; anti-6x His (ab9108) was purchased from Abcam; anti-MyoF (HPA014245) and anti-Myc (06–340) were purchased from MilliporeSigma; anti-HTLV-1 Env gp46 (1C11, sc-53890; 65/6C2.2.34, sc-57865), anti-Gag p19 (TP7, sc-57870) and anti-β-actin (C4, sc-47778) were purchased from Santa-Cruz; anti-clathrin (D3C6, #4796), anti-caveolin-1 (D46G3, #3267), anti-EEA1 (C45B10, #3288), anti-Rab5a (E6N8S, #46449), anti-Rab7 (D95F2, #9367), anti-Rab11 (D4F5, #5589), anti-LAMP-1 (D2D11, #9091) were purchased from Cell Signaling; anti-EHD2 (PA5-80576) was purchased from ThermoFisher Scientific. All blots were developed using Pierce ECL 2 (Thermo Fisher Scientific) and scanned with a Typhoon RGB imager (Cytiva). Immunoblot quantification was performed using ImageQuant TL v8.1 (Cytiva). To enrich Env SU/gp46 when transiently expressed in HEK293T cells, whole cell extracts were immunoprecipitated with Lens Culinary Agglutinin lectin bound to agarose beads (Vector Laboratories) [26 (link)] and beads resuspended in SDS loading dye.
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