Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Alpha-tubulin, clone DM1A | Sigma Aldrich | T9026 Mouse monoclonal RRID:AB_477593 |
IF (1:500) |
| Antibody | Alpha-tubulin-FITC, clone DM1A | Sigma Aldrich |
F2168 Mouse monoclonal RRID:AB_477593 |
IF (1:200) |
| Antibody | Amersham ECL Mouse IgG, HRP-linked F(ab')₂ fragment (from sheep) |
GE Life Sciences |
NA9310 | WB (1:10000) |
| Antibody | Amersham ECL Rabbit IgG, HRP-linked F(ab')₂ fragment (from donkey) |
GE Life Sciences | NA9340 | WB (1:10000) |
| Antibody | Aurora A | Sigma Aldrich | SAB2500135 Goat polyclonal |
WB (1:1000) |
| Antibody | Centromere (ACA) | Antibodies Inc. | 15-234-0001 Human polyclonal |
IF (1:200) |
| Antibody | Phospho-Aurora A (T288), clone C39D8 | Cell Signaling |
3079 Rabbit polyclonal |
IF (1:500); WB (1:1000) |
| Antibody | Donkey anti-goat IgG-HRP |
Santa Cruz | sc-2020 | WB (1:10000) |
| Antibody | Donkey anti-Mouse IgG, Alexa Fluor 488 | Invitrogen | A-21202 | IF (1:500) |
| Antibody | Donkey anti-Mouse IgG, Alexa Fluor 555 | Invitrogen | A-31570 | IF (1:500) |
| Antibody | Donkey anti-Mouse IgG, Alexa Fluor 647 | Invitrogen | A-11126 | IF (1:500) |
| Antibody | Donkey anti-Rabbit IgG, Alexa Fluor 488 | Invitrogen | A-21206 | IF (1:500) |
| Antibody | Donkey anti-Rabbit IgG, Alexa Fluor 555 |
Invitrogen | A-31572 | IF (1:500) |
| Antibody | Donkey anti-Rabbit IgG, Alexa Fluor 647 | Invitrogen | A-31573 | IF (1:500) |
| Antibody | DyLight 405 AffiniPure Donkey Anti-Human IgG (H+L) | Jackson Labs | RRID: AB_2340553 | IF (1:500) |
| Antibody | GAPDH-HRP | Novus | NB110-40405 Mouse monoclonal RRID: AB_669249 |
WB (1:2000) |
| Antibody | Gamma-tubulin | Abcam | 11317 Rabbit polyclonal RRID: AB_297921 |
IF (1:1500) |
| Antibody | Gravin, clone JP74 | Sigma Aldrich |
G3795 Mouse monoclonal |
WB (1:1000) |
| Antibody | Gravin, clone R3698 |
(Nauert et al., 1997) | Rabbit polyclonal |
IF (1:1000) |
| Antibody | Phospho-Gravin (T766) | (Canton et al., 2012) | Rabbit polyclonal |
IF (1:1000) |
| Antibody | Phospho-Hec1 (S69) | (DeLuca et al., 2018) | Rabbit polyclonal |
IF (1:3000) WB (1:3000) |
| Antibody | Phospho-Plk1 (T210) | Biolegend | 628901 Rabbit polycolonal RRID: AB_439786 |
IF (1:500); WB (1:1000) |
| Antibody | Plk1, clone 35–206 | Millipore | 05–844 Mouse monoclonal RRID:AB_11213632 |
IF (1:500); WB (1:1000) |
| Antibody | SNAP-tag | New England Biolabs |
P9310S Rabbit polyclonal |
WB (1:1000) |
| Cell line (H. sapein) | HEK293 Control shRNA stable cell line |
(Canton et al., 2012) | Maintained in Scott lab in DMEM supplemented with 10% FBS under 4 ug/mL Puromycin selection |
|
| Cell line (H. sapein) |
HEK293 Gravin shRNA stable cell line |
(Canton et al., 2012) | Maintained in Scott lab in DMEM supplemented with 10% FBS under under 4 ug/mL Puromycin selection |
|
| Cell line (H. sapein) |
HeLa | From L. Wordeman lab, origin ATCC | Maintained in the Scott Lab in DMEM supplemented with 10% FBS |
|
| Cell line (H. sapein) | hTERT-RPE | Gift from P. Jallepalli lab, origin ATCC |
Maintained in the Scott Lab in DMEM/F-12, Hepes with 10% FBS |
|
| Cell line (M. musculus) |
MEF Gravin wildtype |
(Hehnly et al., 2015) | ||
| Cell line (M. musculus) | MEF Gravin knockout |
(Hehnly et al., 2015) | ||
| Cell line (H. sapein) |
U2OS | ATCC | HTB-96 | Maintained in the Scott Lab in DMEM supplemented with 10% FBS |
| Chemical compound, drug |
Alisertib, MLN8237 | AdooQ | A10004-10nM-D | Manufacturer’s instructions |
| Chemical compound, drug |
BI2536 | AdooQ | A10134-50 | Manufacturer’s instructions |
| Chemical compound, drug |
Beta-mercaptoethanol (BME) | Sigma Aldrich |
M6250 | |
| Chemical compound, drug | CLP-BI2536 | This paper | See ‘synthesis of CLP-reagents’ | |
| Chemical compound, drug |
CLP-MLN8237 | This paper | See ‘synthesis of CLP-reagents’ | |
| Chemical compound, drug |
CLP-rhodamine | This paper | See ‘synthesis of CLP-reagents’ | |
| Chemical compound, drug |
DAPI | Thermo Fisher | 62248 | IF (1:1000) |
| Chemical compound, drug |
Dimethylsulfoxide (DMSO) | Pierce | TS-20688 | Manufacturer’s instructions |
| Chemical compound, drug |
DMEM FluoroBrite | Life Technologies | A1896701 | |
| Chemical compound, drug | DMEM/F-12 Hepes | Life Technologies | 11330057 | |
| Chemical compound, drug |
DMEM, high glucose |
Life Technologies | 11965118 | |
| Chemical compound, drug |
Doxycycline hyclate | Sigma Aldrich | 24390-14-5 | |
| Chemical compound, drug |
Fetal Bovine Serum |
Thermo Fisher |
A3382001 | |
| Chemical compound, drug |
Lipofectamine 2000 Transfection Reagent |
Invitrogen | 11668027 | |
| Chemical compound, drug |
NuPAGE LDS Sample Buffer 4X | Thermo Fisher | NP0008 | |
| Chemical compound, drug |
Opti-MEM I Reduced Serum Medium, no phenol red |
Life Technologies |
11058021 | |
| Chemical compound, drug |
ProLong Diamond Antifade Mountant |
Life Technologies |
P36961 | Manufacturer’s instructions |
| Chemical compound, drug |
Polybrene | Santa Cruz | 134220 | Manufacturer’s instructions |
| Chemical compound, drug |
Puromycin dihydrochloride |
Santa Cruz | 58-58-2 | 4 ug/mL |
| Chemical compound, drug |
SNAP-Cell Fluorescein |
New England Biolabs |
S9107S | Manufacturer’s instructions |
| Chemical compound, drug |
SNAP-Cell 647-SiR | New England Biolabs | S9102S | Manufacturer’s instructions |
| Chemical compound, drug |
SuperSignal West Dura Extended Duration Substrate |
Thermo Fisher |
34075 | |
| Chemical compound, drug |
TransIT-LT1 Transfection Reagent |
Mirus | MIR2300 | |
| Chemical compound, drug |
Trypsin-EDTA (0.25%), phenol red | Gibco | 25200056 | |
| Commercial assay or kit |
BCA Protein Assay Kit |
Thermo Fisher |
23227 | |
| Commercial assay or kit |
QuikChange II XL kit | Aligent | 200522 | |
| Commercial assay or kit |
GeneJET Genomic DNA purification kit |
Thermo Fisher |
K0721 | |
| Peptide, recombinant protein |
Aurora A, active |
Invitrogen | PV3612 | |
| Peptide, recombinant protein |
Plk1, active | SignalChem | P41-10H | |
| Recombinant DNA reagent |
EMTB-3XGFP | Addgene | pCS2+ backbone | |
| Recombinant DNA reagent |
GFP-H2B | Addgene | pEGFP-N1 backbone |
|
| Recombinant DNA reagent |
His6-SNAP-tag | Addgene | pMCSG7 backbone |
|
| Recombinant DNA reagent |
pMD2.G | RRID: Addgene_12259 | gift from Didier Trono; Addgene plasmid #12259 |
|
| Recombinant DNA reagent |
psPAX2 | RRID: Addgene_12260 | gift from Didier Trono; Addgene plasmid #12259 |
|
| Recombinant DNA reagent | SNAP-PACT | This paper | In-house modified pLIX402 backbone (gift from David Root; Addgene plasmid #41394) |
|
| Recombinant DNA reagent |
SNAP-PACT (C144A) | This paper | In-house modified pLIX402 backbone (gift from David Root; Addgene plasmid #41394) |
|
| Recombinant DNA reagent |
SNAP-Mis12 | This paper | In-house modified pLIX402 backbone (gift from David Root; Addgene plasmid #41394) |
|
| Recombinant DNA reagent |
SNAP-Mis12 (C144A) | This paper | In-house modified pLIX402 backbone (gift from David Root; Addgene plasmid #41394) |
|
| Recombinant DNA reagent | SNAP-AKAP79 | This paper | In-house modified pcDNA3.1+ backbone (Life Technologies) |
|
| Recombinant DNA reagent |
SNAP-dAKAP1 | This paper | In-house modified pcDNA3.1+ backbone (Life Technologies) |
|
| Software, algorithm |
Fiji/ImageJ | ImageJ (http://imagej.nih.gov/ij/) | ||
| Software, algorithm | GraphPad Prism |
GraphPad Prism (https://graphpad.com) |
||
| Software, algorithm | Imaris | Bitplane | ||
| Software, algorithm |
SoftWoRx | GE Healthcare | ||
| Other | 1.5 poly-D-lysine coated coverslips | neuVitro | GG-12–1.5-pdl | |
| Other | AnykD Criterion TGX Precast Midi Protein Gel |
Biorad | 5671124 | |
| Other | Bolt 4–12% Bis-Tris Plus Gels | Invitrogen | NW04120BOX | |
| Other | Scienceware cloning discs |
Sigma Aldrich |
Z374431 | |
| Other | μ-Slide 4 Well Glass Bottom: # 1.5H (170 μm + /- 5 μm) D 263 M Schott glass |
(Ibidi)Pierce | 80426 |