Overexpression of TBC1D20 alters the apparent MW of Env and decreases its incorporation into VLPs. (A) HIV-1 VLPs, pseudotyped with JRCSF envelope, were generated in 293T cells (~2 × 106/well, 6-well plate) overexpressing the indicated GAPs (TBC1D20 or R105A) or an empty vector. Cell extracts were prepared from transfected cells and from virion pellets that were purified from the supernatants of the transfected cultures. Western blot was used to detect the expression of Env, Myc-tagged GAPs, CA and GAPDH, using monoclonal antibodies against gp41 [Chessie 8 hybridoma, ; Myc epitope (9E10 hybridoma); CA [183-H12-5C hybridoma, ] and GAPDH (Chemicon, cat. #MAB374). (B) Identical procedure was used to monitor the effect of overexpression of the indicated GAPs on the apparent MW of gp41 (JRFL, LAI and JRCSF strains) and VSV-G; and (C) to monitor this effect on JRCSF Env upon overexpression of the indicated Rab forms. These forms were expressed as GFP fusions , where the parental pEGFP-C1 plasmid (Clonetech), expressing the GFP alone, served as a negative control. Antibodies against GFP (Convance, cat. #MMS-118R) and actin (MP Biomedicals, cat. #69100) were used to detect protein expression.