Co-Immunoprecipitation of APP and HIV-1 Gag

For co-IP, CHME3 cells (3 × 10⁶) were transfected with 2 μg of APP-Flag or HA-tagged forms of HIV-1 Gag alone, or in combination using 15 μl Turbofect (Thermo scientific). Total DNA amount in each group was kept constant with addition of empty vector pcDNA3.1⁻. Soluble cell extracts were prepared 48 h post-transfection as described^{20 (link)} and precleared with protein G-sepharose. A concentration of 27 μl of the input samples was taken and the remainder of the cell extract was incubated with 2 μl of the mouse anti-APP antibody (Invitrogen, 130200) and protein G-sepharose for 1 h at 4 °C. Immune complexes were then washed and boiled with Laemmli buffer and subjected to WB analysis. For co-IP of endogenous APP with Gag, 1 × 10⁶ CHME3 4 × 4 cells were infected with WT HIV-1 or mock virus 48 h before cell lysates were subjected to co-IP as described above. For GBP-binding assay, 293T cells (3 × 10⁶) were transfected with 2 μg of a GFP-expressing control vector (GFP) or N′-GFP-APP₇₇₀ (GFP-APP) along with 2 μg of HA-tagged forms of HIV-1 Gag (Gag-HA, Gag-N-Myr-HA or Gag-MA: -20-HA, -40-HA, -60-HA, and -80-HA) using 15 μl Turbofect (Thermo scientific). Two days post-transfection, cells were lysed with 1 ml cold NP-40 lysis buffer, subjected to GBP-binding assay followed by WB analysis as described in ref. ^{20 (link)}.