Table 1.
Putative binding candidates for NBCn1 C-terminal in MCF-7 cells.
| Gene | Protein ID | C-terminal Intensity 10 6 | C-terminal/GST |
|---|---|---|---|
| Protein sorting machinery | |||
| AP2A1 | AP-2 subunit alpha-1 | 11.0 | 1.3 |
| AP2B1 | AP-2 subunit beta | 9.8 | 3.8 |
| AP2M1 | AP-2 subunit mu | 16.8 | 4.4 |
| CLTA | Clathrin light chain A | 17.4 | — |
| CLTC | Clathrin heavy chain 1 | 774.9 | 2.1 |
| EEA1 | Early endosome | 1.3 | 2.6 |
| Rab5A | Ras-related protein 5A | 42.9 | — |
| Rab5B | Ras-related protein 5B | 10.4 | — |
| Rab5C | Ras-related protein 5C | 173.7 | — |
| Rab8A | Ras-related protein 8A | 40.0 | — |
| Rab11 | Ras-related protein 11 | 188.8 | 24.3 |
| Scaffolding proteins | |||
| GNB2L1 | RACK1 | 205.0 | — |
| CTTN | Cortactin | 7.9 | 1.7 |
| Protein degradation machinery | |||
| LAMP1 | Lysosomal membrane protein 1 | 118.9 | 22.9 |
| LAMP2 | Lysosomal membrane protein 2 | 35.8 | 20.6 |
| UBA1 | Ubiquitin activating enzyme 1 (E1) | 801.9 | 3.7 |
| UBA6 | Ubiquitin activating enzyme 6 (E1) | 343.0 | 46.9 |
| UBE2V1 | Ubiquitin conjugated enzyme (E2) | 229.2 | — |
| UBE2N | Ubiquitin conjugated enzyme (E2) | 280.1 | — |
| SYVN1 | Ubiquitin ligase (E3) | 1.6 | — |
| UBR4 | Ubiquitin ligase (E3) | 26.3 | 7.6 |
| RBX1 | Ubiquitin ligase (E3) | 21.5 | — |
| HUWE1 | Ubiquitin ligase (E3) | 15.6 | 5.4 |
| STUB1 | Ubiquitin ligase (E3) | 52.2 | — |
| Retromer and WASH complex | |||
| SNX1 | SNX1 | 7.3 | 6.1 |
| SNX2 | SNX2 | 35.3 | 10.9 |
| SNX5 | SNX5 | 13.7 | — |
| SNX6 | SNX6 | 44.2 | 13.9 |
| VPS26a | VPS26a | 8.1 | 3.3 |
| VPS26b | VPS26b | 3.6 | — |
| VPS29 | VPS29 | 22.4 | — |
| VPS35 | VPS35 | 149.2 | 18.8 |
| SNX27 | SNX27 | 84.5 | 157.1 |
| KIAA0196 | Strumpellin | 0.9 | 2.3 |
| Polarity complex | |||
| SCRIB | Scribble | 2.2 | — |
| LLGL1 | Lethal giant larvae 1 | 0.96 | — |
Putative binding candidates for NBCn1 C-terminal in MCF-7 cells. The NBCn1-C-tail tagged with GST was produced recombinantly in E. coli. MCF-7 cells were grown to 90% confluency and lysed. NBCn1-C-GST-bound beads were added to the lysates, with GST-only conjugated beads and MCF-7 cell lysates with no beads as control conditions. Proteins were isolated and prepared for mass spectrometry, and data analysed, as described in Materials and Methods. An independent experiment conducted in MCF-7 cells expressing p95HER2 yielded similar results. The values listed in the table are intensities in millions (106). A C-terminal/GST ratio was calculated based on intensities in the C-terminal pull-down and GST-pull-down. “–”, indicates no intensity measured for GST, thus no ratio calculated.