Table 2.
Expression vectors employed in this study.
| Construct | Plasmid | Description | Fragment boundaries (restriction sites) |
Purification | Purpose |
|---|---|---|---|---|---|
| Myc-Spir1-FL | pcDNA3-Myc-hs-Spir1 | Myc-hs-Spir1 | aa 2 - 757 (BamHI / SacI) |
Co-IP | |
| Myc-Spir2-FL | pcDNA3-Myc-hs-Spir2 | Myc-hs-Spir2 | aa 2 - 714 (BamHI / HindIII) |
Co-IP | |
| Myc-Spir2-KWM | pcDNA3-Myc-hs-Spir2- KIND-WH2-GTBM |
Myc-hs-Spir2-KIND- WH2-GTBM |
aa 2 - 437 (BamHI / XhoI) |
Co-IP, Transient expression |
|
| Myc-Spir2-2-423 | pcDNA3-Myc-hs- Spir2-2-423 |
Myc-hs-Spir2-aa2-423 | aa 2 - 423 (BamHI / XhoI) |
Co-IP | |
| Myc-Spir2-KW | pcDNA3-Myc-hs- Spir2-KIND-WH2 |
Myc-hs-Spir2- KIND-WH2 |
aa 2 - 410 (BamHI / XhoI) |
Co-IP, Transient expression |
|
| Myc-Spir2-MSF | pcDNA3-Myc-hs- Spir2-GTBM-SB-FYVE |
Myc-hs-Spir2-GTBM- SB-FYVE |
aa 361/398 - 728 (BamHI / XhoI) |
Co-IP, Transient expression |
|
| Myc-Spir2-411-714 | pcDNA3-Myc-hs- Spir2-411-714 |
Myc-hs-Spir2- aa411-714 |
aa 411 - 714 (BamHI / XhoI) |
Co-IP | |
| Myc-Spir2-SF | pcDNA3-Myc-hs- Spir2-SB-FYVE |
Myc-hs-Spir2- SB-FYVE |
aa 438 - 714 (BamHI / XhoI) |
Co-IP, Transient expression |
|
| Strawberry- Spir2-MSF |
pmStrawberry-C1-hs- Spir2-GTBM-SB-FYVE |
mStrawberry-hs- Spir2-GTBM-SB-FYVE |
aa 361 - 714 (EcoRI / KpnI) |
Transient expression, FLIM-FRET |
|
| Strawberry- Spir2-SF |
pmStrawberry-C2- hs-Spir2-SB-FYVE |
mStrawberry- hs-Spir2-SB-FYVE |
aa 438 - 714 (XhoI / BamHI) |
Transient expression, FLIM-FRET |
|
| Strawberry- Spir2-MSF-LALA |
pmStrawberry-C1- hs-Spir2-GTBM- SB-FYVE-LALA |
mStrawberry-hs- Spir2-GTBM-SB- FYVE-L408A,L409A |
aa 361 - 714 (EcoRI / XbaI) |
Transient expression, FLIM-FRET |
|
| GST-Spir2-MSF | pGex-4T3-hs-Spir2- GTBM-SB-FYVE |
GST-hs-Spir2- GTBM-SB-FYVE |
aa 361 - 714 (BamHI / XhoI) |
GSH-Sepharose 4B, Superdex 200 |
GST-Pulldown |
| His-mCherry- Spir2-linker |
pProEx-HTb-mCherry- hs-Spir2-linker |
His6-mCherry- hs-Spir2-linker |
aa 361 - 519 (XhoI / HindIII) |
Ni-NTA, Superdex 200 |
GST-Pulldown, Binding assays |
| His-mCherry- Spir2-linker-LALA |
pProEx-HTb-mCherry- hs-Spir2-linker-LALA |
His6-mCherry-hs-Spir2- linker-L408A,L409A |
aa 361-519 (mutagenesis) |
Ni-NTA, Superdex 200 |
GST-Pulldown |
| Spir2 / Fluorescein- Spir2 peptide |
aa 401-427 | purchased from GenScript |
Crystallization, Fluoresc. anisotropy |
||
| GFP-MyoVa-D-FL | provided by Bruno Goud |
Lindsay et al., 2013 | GST-Pulldown, Transient expression |
||
| GFP-MyoVa-D-QR | provided by Bruno Goud |
Lindsay et al., 2013 | GST-Pulldown, Transient expression |
||
| GFP-MyoVa-F-QR | provided by Bruno Goud |
Lindsay et al., 2013 | Transient expression | ||
| GFP-MyoVa- CC-GTD |
peGFP-C2- mm-MyoVa-CC-GTD |
eGFP-mm-MyoVa- CC-GTD |
aa 1260 - 1880 (EcoRI / SalI) |
Transient expression | |
| GFP-MyoVa-GTD | pAcGFP-C1- mm-MyoVa-GTD |
AcGFP-mm- MyoVa-GTD |
aa 1467 - 1880 (HindIII / SalI) |
Co-IP, FLIM-FRET |
|
| Strawberry- MyoVa-GTD |
pmStrawberry-C1- mm-MyoVa-GTD |
mStrawberry-mm- MyoVa-GTD |
aa 1467 - 1880 (HindIII / SalI) |
Transient expression, FLIM-FRET |
|
| GST-MyoVa-GTD | pGex-4T1-NTEV- mm-MyoVa-GTD |
GST-mm-MyoVa-GTD | aa 1467 - 1880 (HindIII / SalI) |
GSH-Sepharose 4B, Superdex 200 |
GST-Pulldown, Binding assays |
| MyoVa-GTD | pProEx-HTb-hs- MyoVa-GTD |
hs-MyoVa-GTD | aa 1461 - 1853 (BamHI / XhoI) |
HisTrap, TEV, Superdex 200 |
Crystallization, GST- Pulldown, Anisotropy |
| GFP-MyoVb- CC-GTD |
pAcGFP-C1- hs-MyoVb-CC-GTD |
AcGFP-hs- MyoVb-CC-GTD |
aa 904 - 1848 (HindIII / SalI) |
Co-IP | |
| GFP-MyoVb-GTD | pAcGFP-C1- hs-MyoVb-GTD |
AcGFP-hs- MyoVb-GTD |
aa 1467 - 1848 (HindIII / SalI) |
Co-IP, FLIM-FRET, Transient expression |
|
| GFP-MyoVb-GTD- Q1748R |
pAcGFP-C1-hs- MyoVb-GTD-Q1748R |
AcGFP-hs-MyoVb- GTD-Q1748R |
aa 1467 - 1848 (mutagenesis) |
Transient expression | |
| GST-MyoVb-GTD | pGex-4T1-NTEV- hs-MyoVb-GTD |
GST-hs-MyoVb-GTD | aa 1467 - 1848 (BamHI / XhoI) |
GSH-Sepharose 4B, Superdex 200 |
GST-Pulldown, Binding assays |
| MyoVb-GTD | pProEx-HTb-hs- MyoVb-GTD |
hs-MyoVb-GTD | aa 1456 - 1848 (BamHI / XhoI) |
HisTrap, TEV, Superdex G200 |
Crystallization, GST- Pulldown, Anisotropy |
| MyoVc-GTD | pProEx-HTb-hs- MyoVc-GTD |
hs-MyoVc-GTD | aa 1350 - 1742 (BamHI / XhoI) |
HisTrap, TEV, Superdex 200 |
Microscale thermophoresis |
| Strawberry- Rab11a |
pmStrawberry-C1- mm-Rab11a |
mStrawberry- mm-Rab11a |
aa 2 - 216 (EcoRI / BamHI) |
Transient expression | |
| GST-Rab11a-Q70L | pGex-4T1-NTEV- cl-Rab11a-Q70L |
GST-cl-Rab11a-Q70L | aa 1 - 216 (EcoRI / EcoRI) |
GSH-Sepharose 4B, Superdex 200 |
GST-Pulldown |
| Rab11a | pET28-rTEV- hs-Rab11a |
hs-Rab11a | aa 1 - 177 (NcoI / EcoRI) |
HisTrap, TEV, Superdex 200 |
Crystallization, SPR |
| Rab11a-Q70L | pET28-rTEV- hs-Rab11a-Q70L |
hs-Rab11a-Q70L | aa 1 - 173 (NcoI / EcoRI) |
HisTrap, TEV, Superdex 200 |
GST-Pulldown |
| Strawberry-FMN2- FH2-FSI |
pmStrawberry-C2- mm-FMN2-FH2-FSI |
mStrawberry-mm- FMN2-FH2-FSI |
aa 1135 - 1578 (BamHI / XhoI) |
GST-Pulldown | |
| GST-FMN2-eFSI | pGex-4T1-NTEV- mm-FMN2-eFSI |
GST-mm-FMN2-eFSI | aa 1523 - 1578 (BamHI / XhoI) |
GSH-Sepharose FF, Superdex 200 |
GST-Pulldown from brain lysates |
| GFP | pAcGFP-C1 | AcGFP | Co-IP, FLIM-FRET |
||
| GFP-linker- Strawberry |
pAcGFP-C1-linker- mStrawberry |
AcGFP-linker(A-S- G-A-G)-mStrawberry |
aa 1 - 236 (BspEI / BglII) |
FLIM-FRET | |
| mCherry | pProEx-HTb-mCherry | His6-mCherry | aa 1 - 236 (BamHI / XhoI) |
Ni-NTA, Superdex 200 |
GST-Pulldown |
| GST | pGex-4T1-NTEV | GST | GSH-Sepharose 4B, Superdex 200 |
GST-Pulldown |