TABLE 1.
Manders’ colocalization coefficients determined in this studya
| Proteins | Mean Manders’ coefficient ± SEM (no. of cells) |
|
|---|---|---|
| tM1 | tM2 | |
| HA-HLA-A2 and TGN46 in HeLa cells (siRNA control) | 0.09 ± 0.009 (5) | 0.21 ± 0.07 (5) |
| HA-HLA-A2 and TGN46 in HeLa cells expressing Nef (siRNA control) | 0.39 ± 0.02 (8) | 0.45 ± 0.03 (8) |
| HA-HLA-A2 and TGN46 in HeLa cells expressing Nef (siRNA AP-1γ1) | 0.16 ± 0.01 (6) | 0.19 ± 0.03 (6) |
| HA-HLA-A2 and TGN46 in HeLa cells expressing Nef (siRNA AP-1γ2) | 0.16 ± 0.006 (8) | 0.26 ± 0.02 (8) |
| HLA-A and AP-1γ1 in A3.01 GFP T cells (shRNA control) | 0.17 ± 0.02 (6) | 0.24 ± 0.01 (6) |
| HLA-A and AP-1γ1 in A3.01 Nef/GFP T cells (shRNA control) | 0.77 ± 0.005 (7) | 0.63 ± 0.01 (7) |
| HLA-A/B/C and AP-1γ2 in A3.01 GFP T cells (shRNA control) | 0.20 ± 0.02 (6) | 0.20 ± 0.02 (6) |
| HLA-A/B/C and AP-1γ2 in A3.01 Nef/GFP T cells (shRNA control) | 0.73 ± 0.03 (7) | 0.45 ± 0.02 (7) |
| HLA-A and TGN46 in A3.01 GFP T cells (shRNA control) | 0.31 ± 0.02 (5) | 0.44 ± 0.03 (5) |
| HLA-A and TGN46 in A3.01 Nef/GFP T cells (shRNA control) | 0.64 ± 0.01 (5) | 0.64 ± 0.01 (5) |
| HLA-A and TGN46 in A3.01 Nef/GFP T cells (shRNA AP-1γ1) | 0.33 ± 0.02 (5) | 0.44 ± 0.01 (5) |
| HLA-A and TGN46 in A3.01 Nef/GFP T cells (shRNA AP-1γ2) | 0.38 ± 0.01 (5) | 0.45 ± 0.02 (5) |
| AP-1γ2 and AP-1γ1 in GFP T cells | 0.57 ± 0.02 (6) | 0.79 ± 0.01 (6) |
| AP-1γ2 and AP-1γ1 in Nef/GFP T cells | 0.28 ± 0.02 (6) | 0.65 ± 0.03 (6) |
The colocalization threshold plug-in for ImageJ software (55) was used for the quantification of colocalization. For the determination of single-channel-specific Manders’ overlap coefficients, tM1 is defined as the total intensity of pixels from channel 1 (green) for which the intensity in channel 2 (red) is above zero relative to the total intensity in channel 1, and tM2 is defined as the total intensity of pixels from channel 2 for which the intensity in channel 1 is above zero relative to the total intensity in channel 2. The coefficients were calculated using an automatically determined threshold value, for both channels, according to an algorithm described previously (56), where a value of 1 represents total colocalization. Shown are the means ± standard errors of the means (SEM) for tM1 and tM2, indicating the extent of the overlap between the signals of the indicated proteins. Data for each pairwise comparison were obtained from a series of z-slices (with 0.3-μm intervals) for at least five cells from three independent experiments.