Fig. 5

ATG9A is dispensable for SERINC5 downregulation by Nef. a WT and ATG9A-KO Jurkat cells transiently expressing SERINC5-GFP minus (−) or plus (+) Nef-mCherry were fixed, permeabilized, and imaged by confocal microscopy. Scale bars: 10 μm. b Lysates of WT or ATG9A-KO Jurkat cells, transiently expressing mCherry (−) or HIV-1 Nef-mCherry (+) in addition to SERINC5-GFP, were subjected to SDS-PAGE and immunoblotting with antibodies to ATG9A, mCherry, GFP or actin. The positions of molecular mass markers (in kDa) are indicated on the left. The different bands detected by the GFP antibody correspond to different glycosylation forms of SERINC5 [17]. c WT and ATG9A-KO HeLa cells were co-transfected with 50 ng of SERINC5 and 1 μg of WT or ΔNef pNL4-3 for 6–8 h and the medium was replaced. One day later, the culture supernatant was filtered, and virus pellets were collected by ultracentrifugation. Cell and virus lysates were analyzed by SDS-PAGE and immunoblotting with HIV-Ig to detect viral proteins (Pr55^Gag, CAp24), and with antibodies to HIV-1 Nef, ATG9A and tubulin. In b and c, the positions of molecular mass markers (in kDa) are indicated on the left. d Infectivity of virions was measured in TZM-bl cells as in Fig. 3. Values were normalized to the infectivity of WT HIV-1 from parental HeLa cells in the absence of SERINC5. Data were evaluated for statistical significance by using a one-way ANOVA followed by a Holm-Šídák post hoc test; ns: not significant (p > 0.05), *p < 0.05, ****p < 0.0001)