FIG 7.

Nonimmune controls for the biotin-Gag, biotin-ABCE1, and biotin-DDX6 proximity ligation assays. (A) NI controls for the biotin-Gag PLA. Above each PLA image is shown the schematic corresponding to the PLA approach in that panel. 293T cells chronically infected with HIV-1 (293T-HIV) were treated with 10 μM PAV-818 (the biotinylated active compound) for 16 h. For the positive control (image and schematic on the right), PLA was performed by incubation with primary antibodies, mouse anti-Gag and rabbit anti-biotin, followed by PLA secondary antibodies and other reagents as described in Fig. 6. In the two negative controls (image and schematic at left and center), one primary antibody was replaced with a NI control antibody from the same species, as indicated. Red spots indicating colocalization of the biotinylated compound with Gag should be absent when either primary antibody is replaced by a NI antibody. After PLA, IF was performed to mark either Gag-expressing or biotin-containing cells with low-level green fluorescence. Images show a representative field for each of the three antibody pairs. Fields are shown as a merge of three color channels: the red channel shows biotin-NI PLA, NI-Gag PLA, or biotin-Gag PLA, as indicated by red labeling above images; the green channel shows biotin IF or Gag IF, as indicated by green labeling above images; and the blue channel shows DAPI-stained nuclei. Scale bars, 10 μm. Graph below shows the average number of PLA spots per cell for each antibody pair. A total of 10 to 20 fields were analyzed for each group (containing 118 to 213 cells per group), with error bars showing the SEM. (B) NI controls for the biotin-ABCE1 PLA (top row of images) and biotin-DDX6 PLA (bottom row of images). Figure organization and 293T-HIV treatments are as in panel A above. For the positive control (images and schematic on the right), PLA was performed by incubation with primary antibodies, rabbit anti-host protein (ABCE1 in top row; DDX6 in bottom row) and mouse anti-biotin, followed by PLA secondary antibodies and other reagents as described in Fig. 6. In the two negative controls (images and schematics at left and center), one primary antibody was replaced with a NI control antibody from the same species, as indicated. Red spots indicating colocalization of the biotinylated compound with the host proteins ABCE1 and DDX6 should be absent when either primary antibody is replaced by a NI antibody. After PLA, IF was performed with the indicated antibody (green fluorescence). Images show a representative field for each of the three antibody pairs. Fields are shown as a merge of three color channels: the red channel shows biotin-NI PLA, NI-host protein PLA, or biotin-host protein PLA, as indicated by red labeling above images; the green channel shows biotin IF or host protein IF, as indicated by green labeling above images; and the blue channel shows DAPI-stained nuclei. Scale bars, 10 μm. Graphs below shows the average number of PLA spots per cell for each antibody pair (ABCE1 on the left; DDX6 on the right). Five fields were analyzed for each group (containing a total of 43 to 77 cells per group), with error bars showing the SEM.