Fig. 2. Quantitative analysis of α4β7 incorporation by HIV-1 virions.

Western blot analysis of α4 and HIV-1 structural proteins (gp120_Env and p24_Gag) incorporated into HIV-1 virions produced by RA-treated human PBMC (RA) vs. untreated human PBMC (Unt.). Virus stocks were concentrated by PEG treatment and centrifugation before detergent lysis. As a control for integrin subunit expression on producer cells, we loaded whole cell lysate from RA-treated CD4⁺ T cells. For semi-quantitative assessment of virion incorporation, we used serial dilutions of recombinant α4β7 and recombinant gp120, respectively, at the indicated amounts loaded per lane (in nanograms). The different molecular weight of recombinant α4 (120 kDa) vs. cell-derived α4 (70kDa) reflect the fact that activated T cells express cleaved α4 (α4_cl) on their surface, while recombinant α4 is uncleaved (α4_un). Semi-quantification of incorporated α4β7 and gp120 was performed by densitometric scanning and interpolation of the results on the curves obtained for the recombinant proteins used as reference. The gel was also developed using pooled sera from HIV-infected individuals (bottom panel) to verify the correct amount of input p24G_ag antigen. This Western blot is representative of two experiments performed with similar results.