Fig. 2

HIV-1 Nef and Vpu downregulate total CD28 protein levels. CD4⁺ Sup-T1 cells or primary CD4⁺ T cells were infected with VSV-G pseudotyped NL4.3. Viruses encoded Nef and Vpu (NL4.3, red), lacked Nef (dNef, blue), lacked Vpu (dVpu, orange) or lacked both Nef and Vpu (dNef dVpu, green). Forty-eight hours post-infection, cells were permeabilized, stained for CD28 and analyzed by flow cytometry. a Representative dot plots illustrating total CD28 (APC) and infection (GFP) levels of live (Zombie Red^TM−) Sup-T1 cells. b Representative histograms illustrating total levels of CD28 (APC) after gating on live (Zombie Red^TM−) infected (GFP⁺) cells. Geometric mean fluorescence intensities (MFI) are indicated. c Summary of mean (± SE) relative total CD28 based on MFI (n = 12). d Representative dot plots illustrating purified CD4⁺ T cell infection (GFP) and total CD28 (APC) levels. e Summary of mean (± SE) total CD28 levels on infected (GFP⁺) CD4⁺ T cells (n = 5). Data were obtained from infection of cells obtained from two healthy donors. (UI: uninfected; MFI: geometric mean fluorescent intensity; SE: standard error; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)