TABLE 1.
Tac chimera trafficking to and steady-state localization at the TGN in TRVb1/TTF and TRVb1/Tac-TGN38 cells Cells scored for “perinuclear cluster” displayed the typical high focal concentration of Tac-associated immunofluorescence at the perinuclear TGN. Under conditions indicated by an asterisk Tac-furin was distributed predominantly in peripheral cytoplasmic punctae, but not at the perinuclear cluster, i.e. the TGN, as illustrated in Figs. 5 and 6. Quantification is based on counting at least 100 cells/condition in two to four coverslips (mean ± S.E.; *, p < 0.001 vs. respective controls).
|
Tac-Furin
|
Tac-TGN38
|
|||||
|---|---|---|---|---|---|---|
| Cell treatment | Trafficking perinuclear clustera | Steady-state localization perinuclear clusterb | Trafficking perinuclear clustera | Steady-state localization perinuclear clusterb | ||
| % cells | % cells | |||||
| siRNA-mediated protein knockdown | ||||||
| Cyclophilin (control) | 93 ± 4 | 94 ± 6 | 95 ± 6 | 96 ± 6 | ||
| PIKfyve | 12 ± 3* | 92 ± 5 | 90 ± 5 | 92 ± 5 | ||
| JLP | 15 ± 2* | 91 ± 6 | 94 ± 6 | 93 ± 2 | ||
| Expression of GFP-based constructs | ||||||
| GFP (control) | 90 ± 2 | 95 ± 4 | 94 ± 2 | 95 ± 4 | ||
| PIKfyve-(616–868) | 23 ± 3* | 93 ± 3 | 96 ± 3 | 93 ± 3 | ||
| JLPL-(963–1320) | 27 ± 4* | 94 ± 3 | 93 ± 2 | 94 ± 3 | ||
| PIKfyveK1831E | 22 ± 2* | 94 ± 4 | 94 ± 4 | 94 ± 4 | ||
| PIKfyveWT | 92 ± 2 | 96 ± 4 | 98 ± 5 | 94 ± 4 | ||
| JLPLWT | 90 ± 4 | 92 ± 4 | 92 ± 4 | 92 ± 4 | ||
| Microinjection | ||||||
| GST (control) | 92 ± 2 | 96 ± 3 | 95 ± 3 | 93 ± 4 | ||
| GST-JLPL-(1246–1320) | 15 ± 4* | 92 ± 4 | 91 ± 4 | 96 ± 4 | ||
Non-permeabilized TRVb1/TTF and TRVb1/Tac-TGN38 cells were analyzed for Tac-furin and Tac-TGN38 trafficking, respectively, as described in the legends for Figs 5 and 6
Cells were permeabilized, and Tac chimera steady-state localization was determined with anti-Tac as described under “Experimental Procedures”