Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | HEK293T | ATCC | ATCC Cat# CRL-3216, RRID:CVCL_0063 | |
| Cell line (human) |
Flp-In T-Rex-293 | Thermo Fisher Scientific | RRID:CVCL_U427 | |
| Cell line (human) |
Flp-In T-Rex-HeLa | other | generated by Elena Dobrikova and Matthias Gromeier, Duke University Medical Center, Durham, NC, USA |
|
| Cell line (human) |
HeLa | ATCC | ATCC Cat# CRM-CCL-2, RRID:CVCL_0030 | |
| Recombinant DNA reagent |
pEGFP-N1-PKD1 wt/K612W |
DOI: 10.1038/ncb1289 | ||
| Recombinant DNA reagent |
pEGFP-N1-PKD2 | DOI: 10.1038/ncb1289 | ||
| Recombinant DNA reagent |
pCMV5-EGFP-GEF -H1 wt |
DOI: 10.1038/ncb773 | ||
| Recombinant DNA reagent |
pCMV5-EGFP-GEF -H1 C53R |
DOI: 10.1038/ncb773 | ||
| Recombinant DNA reagent |
pTriEx-RhoA FLARE.sc |
DOI: 10.1038/nature04665 | Addgene #12150 | |
| Recombinant DNA reagent |
mRuby-Golgi-7 | Addgene #55865 | ||
| Recombinant DNA reagent |
Rab6a-GFP | other | provided by Francis Barr, Oxford University |
|
| Recombinant DNA reagent |
DsRed2-Paxillin | DOI: 10.1038/ncb1094 | ||
| Recombinant DNA reagent |
G-PKDrep | doi: 10.1111/j.1600– 0854.2009.00918.x |
||
| Recombinant DNA reagent |
Clip170-GFP | other | provided by Niels Galjart, Erasmus Medical Center, Rotterdam |
|
| Recombinant DNA reagent |
pcDNA5/FRT/TO-EGFP -GEF-H1 wt/C53R |
this paper | ||
| Recombinant DNA reagent |
pECFP-Endo/RhoB | Clontech | Clontech #6934–1 | |
| Recombinant DNA reagent |
pECFP-Endo/Rhob Q63L |
this paper | site-directed mutagenesis using pECFP-RhoB as a template |
|
| Recombinant DNA reagent |
pEGFP-RhoB | this paper | subcloned from pECFP-Endo/RhoB |
|
| Recombinant DNA reagent |
pEGFP-RhoB Q63L | this paper | subcloned from pECFP-Endo/RhoB Q63L |
|
| Recombinant DNA reagent |
pEGFP-RhoA | DOI: 10.1242/jcs.163857 | ||
| Recombinant DNA reagent |
pEGFP-RhoA Q63L | DOI: 10.1242/jcs.163857 | ||
| Recombinant DNA reagent |
pcDNA3.1-HA-RhoA Q63L |
DOI: 10.1242/jcs.163857 | ||
| Recombinant DNA reagent |
pcDNA3.1-HA-RhoA | DOI: 10.1242/jcs.163857 | ||
| Recombinant DNA reagent |
Str-KDEL-TNFa-SBP- mCherry |
Addgene #65279 | ||
| Recombinant DNA reagent |
pOG44 | Thermo Fisher Scientific | Thermo Fisher Scientific V600520 | |
| Antibody | p230 | BD Biosciences | BD Biosciences Cat# 611280, RRID:AB_398808 | |
| Antibody | GFP | Roche | Sigma-Aldrich Cat# 11814460001, RRID:AB_390913 |
|
| Antibody | TGN46 | Bio-Rad/AbD Serotec | Bio-Rad/AbD Serotec Cat# AHP500, RRID:AB_324049 |
|
| Antibody | alpha-tubulin | Millipore | Millipore Cat# 05–829, RRID:AB_310035 |
|
| Antibody | DLC3 | Santa Cruz Biotechnology | Santa Cruz Biotechnology Cat# sc-166725, RRID:AB_2197829 |
|
| Antibody | PKD1 | Cell Signaling | Cell Signaling Technology Cat# 2052, RRID:AB_2268946 |
|
| Antibody | PKD2 | Cell Signaling | Cell Signaling Technology Cat# 8188S, RRID:AB_10829368 |
|
| Antibody | PKD3 | Cell Signaling | Cell Signaling Technology Cat# 5655S, RRID:AB_10695917 |
|
| Antibody | GEF-H1 | Cell Signaling | Cell Signaling Technology Cat# 4076, RRID:AB_2060032 |
|
| Antibody | phospho-PKD (Ser744/748) |
Cell Signaling | Cell Signaling Technology Cat# 2054S, RRID:AB_2172539 |
|
| Antibody | Rab8 | Cell Signaling | Cell Signaling Technology Cat# 6975S, RRID:AB_10827742 |
|
| Antibody | Rab6 | Cell Signaling | Cell Signaling Technology Cat# 9625S, RRID:AB_10971791 |
|
| Antibody | pS910 | DOI: 10.1083/jcb.200110047 | ||
| Antibody | ERK1/2 | Cell Signaling | Cell Signaling Technology Cat# 9107S, RRID:AB_10695739 |
|
| Antibody | MEK1/2 | Cell Signaling | Cell Signaling Technology Cat# 8727, RRID:AB_10829473 |
|
| Antibody | pERK1/2 (Thr202/Tyr204) |
Cell Signaling | Cell Signaling Technology Cat# 4094S, RRID:AB_10694057 |
|
| Antibody | pMEK1/2 (Ser2017/221) |
Cell Signaling | Cell Signaling Technology Cat# 9154, RRID:AB_2138017 |
|
| Antibody | ROCK1 | Millipore | Millipore Cat# 04–1121, RRID:AB_1977472 |
|
| Antibody | ROCK2 | BD Biosciences | BD Biosciences Cat# 610623, RRID:AB_397955 |
|
| Antibody | pS294 | DOI: 10.1038/ncb1289 | IF 1:750 | |
| Antibody | HRP goat anti-rabbit | Dianova | Jackson ImmunoResearch Labs Cat# 111-035-144, RRID:AB_2307391 |
|
| Antibody | HRP goat anti-mouse | Dianova | Jackson ImmunoResearch Labs Cat# 115-035-062, RRID:AB_2338504 |
|
| Antibody | Alexa Fluor labelled secondary antibodies |
Thermo Fisher Scientific | Thermo Fisher Scientific | |
| Sequence-based reagent |
ON-Targetplus smartpools |
Dharmacon | Dharmacon | |
| Sequence-based reagent |
Silencer select siPLCe |
Thermo Fisher Scientific | Thermo Fisher Scientific s27660 |
|
| Sequence-based reagent |
Silencer select siGEF-H1 |
Thermo Fisher Scientific | Thermo Fisher Scientific s17546 |
|
| Sequence-based reagent |
Quantitect primers for RT-PCR |
Qiagen | Qiagen | |
| Peptide, recombinant protein |
Trypsin | Thermo Fisher Scientific | Thermo Fisher Scientific 15090046 |
|
| Peptide, recombinant protein |
Thrombin | Millipore | Millipore 605195 | |
| Commercial assay or kit |
QuantiTect SYBR Green RT-PCR Kit |
Qiagen | Qiagen 204243 | |
| Commercial assay or kit |
Rneasy plus Kit | Qiagen | Qiagen 74104 | |
| Chemical compound, drug |
Nocodazole | Sigma-Aldrich | Sigma-Aldrich M1404 | |
| Chemical compound, drug |
CRT0066101 | Tocris Bioscience | Tocris Bioscience 4975 | |
| Chemical compound, drug |
H1152 | Enzo Life Science | Enzo Life Science ALX-270–423 M001 |
|
| Chemical compound, drug |
UO126 | Cell Signaling | Cell Signaling #9903 | |
| Chemical compound, drug |
Blasticidin | Thermo Fisher Scientific | Thermo Fisher Scientific R21001 |
|
| Chemical compound, drug |
Hygromycin B | Thermo Fisher Scientific | Thermo Fisher Scientific 10687010 |
|
| Chemical compound, drug |
Doxycyclin | Sigma-Aldrich | Sigma-Aldrich D9891 | |
| Chemical compound, drug |
Zeocin | Thermo Fisher Scientific | Thermo Fisher Scientific R25001 |
|
| Chemical compound, drug |
Collagen R | Serva | Serva 47254 | |
| Chemical compound, drug |
Biotin | Sigma-Aldrich | Sigma-Aldrich B4501 | |
| Chemical compound, drug |
Blocking reagent | Roche | Roche 11096176001 | |
| Software, algorithm | Cell Profiler | http://cellprofiler.org | CellProfiler Image Analysis Software, RRID:SCR_007358 |
|
| Software, algorithm | GraphPad Prism | GraphPad Prism (https://graphpad.com) | Graphpad Prism, RRID:SCR_002798 |
|
| Software, algorithm | Image Studio Lite 4.0 |
https://www.licor.com/bio/products/software/image_studio_lite/?utm_source=BIO+Blog&utm_medium=28Aug13post&utm_content=ISLite1&utm_campaign=ISLite | Image Studio Lite, RRID:SCR_014211 |