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. 2013 Mar 11;210(3):517–534. doi: 10.1084/jem.20120572

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Figure 7. — SPTBN1 associates with HIV-1 gag proteins. (A) 293T cells were transfected to express FLAG-tagged SPTBN1 and gag p55. Whole-cell lysates from transfected cells were subjected to a pull-down assay using anti-FLAG agarose. Unrelated FLAG-tagged RIG-I and untagged SPTBN1 were negative controls for nonspecific binding. Immunoprecipitates were analyzed by immunoblotting using anti-FLAG tag and anti-gag p55 antibodies. (B) 293T cells were transfected to express FLAG-tagged SPTBN1 and HA-tagged CA p24, MA p17, and NC p7. Whole-cell lysates from transfected cells were subjected to a pull-down assay using anti-FLAG agarose or anti-HA agarose. Immunoprecipitates were analyzed by immunoblotting using anti-SPTBN1 and anti-HA antibodies. Nef was a negative control for nonspecific binding. (C) Macrophages were transfected to express GFP-tagged Gag p55 (green). After 24 h, macrophages were fixed and labeled with antibody against SPTBN1 (red). A representative merged image of Gag p55 and SPTBN1 shown on the right (yellow) indicates the colocalization between Gag and SPTBN1. R, Pearson coefficient of correlation. Bars, 10 µm. (D) Macrophages were infected with VSV-G–pseudotyped Vpr-GFP-packaged HIV-1 virions (green). After 30-min incubation, cells were fixed and labeled with antibody against SPTBN1 (red) or DAPI (blue). White arrows on the merged image indicate the colocalization of SPTBN1 and incoming HIV-1 virions (yellow). Bars, 30 µm. (E) M-Mac and I-Mac were infected with VSV-G-pseudotyped Vpr-GFP-packaged HIV-1 virions (green). After 30-min incubation, cells were fixed and labeled with antibody against SPTBN1 (red) or DAPI (blue). White arrows on the merged image indicate the colocalization of SPTBN1 and incoming HIV-1 virions (yellow). Bars, 30 µm.