Table 1.
Lists of reagents used in experiments
Cell lines and reagentsCompanyCatalog number and sequences
Cell lines   
HPAECs Lonza cc-2530 (lot numbers 329447, 447095, 466719, 598033, 662151, 4F3034) 
CHO-K1 ATCC TCC CCL-61 
Antibodies   
Phosphotyrosine antibody (PY20) Abcam ab16389 
Phosphotyrosine antibody, clone 4G10 EMD Millipore 05-321X 
VE-cadherin antibody Santa Cruz sc-6458 
Phospho-VE-cadherin (Tyr685) EMD Millipore ABT1760 
Phospho-VE-cadherin (Tyr658) polyclonal antibody ThermoFisher  44-1144G 
Phospho-VE-cadherin (Tyr685) polyclonal antibody Abcam ab119785 
Phospho-VE-cadherin (Tyr731) Polyclonal antibody ThermoFisher  44-1145G 
TIE2 antibody Abcam ab58302 
human/mouse phospho-Tie-2 (Y992) antibody R&D Systems AF2720 
Cingulin polyclonal antibody Novus Biologicals NBP1-89602 
GEF-H1 antibody Abcam ab155785 
Anti–GEF-H1 antibody [B4/7] Abcam ab90783 
p114RhoGEF (ARHGEF18) monoclonal antibody Novus Biologicals NBP2-43546 
p115RhoGEF (D-11; ARHGEF1) monoclonal antibody Santa Cruz sc-166341 
RGNEF (ARHGEF28) polyclonal antibody Abcam ab157095 
GST antibody (A-6) Santa Cruz sc-374171 
6x-His Tag monoclonal antibody ThermoFisher 4E3D10H2/E3 
GFP Tag monoclonal antibody ThermoFisher  GF28R 
PTPβ antibody (C-20) Santa Cruz sc-1114 
RPTPβ antibody BD Biosciences 610180 
Alexa Fluor 488 Phalloidin Invitrogen A12379 
Mouse monoclonal anti-β-tubulin Sigma Aldrich T8328 
Anti-mouse IgG (Fc) antibody Aviva Systems Biology OARD00003 
Primers   
pNH-TRXT-VE-PTP aa 1,651–1,998-6xHis Integrated DNA Technologies VE-PTP aa 1,651 EcorRI Fwd; 5′-ATA​GGA​GAA​TTC​GCC​ACC​ATG​AGG​GAA​AGG​CCA​TCT​GCC-3′ 
VE-PTP aa 1,998 HindIII Rev; 5′-AAA​CCT​AAG​CTT​ATG​TCT​CGA​GTA​GAT​TGC-3′ 
mPlum VE-PTP 1,422–1,998 D1871A Integrated DNA Technologies VE-PTP D1871A Fwd 5′-GCC​AGC​CCA​TGG​GGT​CCC​AGA​G-3′ 
VE-PTP D1871A Rev 5′-CCA​TGG​GCT​GGC​CAC​ACC​GTG​T-3′ 
mPlum VE-PTP 1,611–1,998 Integrated DNA Technologies VE-PTP 1,611 BspEI Fwd 5′-AGC​GCG​TCC​GGA​GCC​ACC​ATG​ACC​ACA​GAG​TCA​GAG​CCC​TTG​TTT​GGA​G-3′ 
VE-PTP 1,998 XhoI Rev 5′-GCC​GCC​TCT​AGA​CTA​ATG​TCT​CGA​GTA​GAT​TGC​ATC​TCT​GTG​ATA​C-3′ 
pAmCyan VE-PTP 1,651–1,998 Integrated DNA Technologies VE-PTP 1,651 BspEI Fwd 5′-GAC​ACG​TCC​GGA​GCC​ACC​ATG​AGG​GAA​AGG​CCA​TCT​GCC​CGG​CTC​AGC-3′ 
VE-PTP 1,998 XhoI Rev 5′-GGG​CCC​CTC​GAG​TTA​ATG​TCT​CGA​ATA​GAT​TGC​ATC​TCT​GTG​ATA​CTC-3′ 
mPlum VE-PTP 1–1,650 Integrated DNA Technologies VE-PTP 1 BspEI 5′ Fwd 5′-AGC​GCG​TCC​GGA​GCC​ACC​ATG​CTG​AGG​CAT​GGA​GCC​CTA​ACG​GCC​TTG-3′ 
VE-PTP 1,650 XhoI Rev 5′-GCC​GCC​CTC​GAG​GCT​AGC​TTT​CTG​TCT​GCA​GAT​GAA​GAA​GGC-3′ 
GST-GEFH1 572–876 Integrated DNA Technologies GEFH1 572 EcoRI Fwd 5′-GGA​CTC​GAA​TTC​GAG​CCA​CCA​TGA​CAT​GCC​CAT​CCA​GGG​AGG​AC-3′ 
GEFH1 876 HindIII Rev 5′-AGG​TCC​AAG​CTT​TTA​ATC​CAC​AGG​TCT​GCG​GGC​CCA-3′ 
GST-GEFH1 731–985 Integrated DNA Technologies GEFH1 731 EcoRI Fwd 5′-GGA​CTC​GAA​TTC​GAG​CCA​CCA​TGA​GAT​CAC​CGC​AAG​AGG​AGG​CG-3′ 
GEFH1 985 HindIII Rev 5′-AGG​TCC​AAG​CTT​TTA​GCT​CTC​GGA​GGC​TAC​AGC​CTC-3′ 
GST-GEFH1 731–876 Integrated DNA Technologies GEFH1 731 EcoRI Fwd 5′-GGA​CTC​GAA​TTC​GAG​CCA​CCA​TGA​GAT​CAC​CGC​AAG​AGG​AGG​CG-3′ 
GEFH1 876 HindIII REV 5′-AGG​TCC​AAG​CTT​TTA​ATC​CAC​AGG​TCT​GCG​GGC​CCA-3′ 
GST-GEFH1 731–925 Integrated DNA Technologies GEFH1 731 EcoRI Fwd 5′-GGA​CTC​GAA​TTC​GAG​CCA​CCA​TGA​GAT​CAC​CGC​AAG​AGG​AGG​CG-3′ 
GEFH1 925 HindIII Rev 5′-AGG​TCC​AAG​CTT​TTA​CCC​CAG​TTC​CTG​CCT​CTC​TCG-3′ 
pAmCyan-VE-PTP 1,611–1,998 Integrated DNA Technologies VE-PTP 1,611 BspEI Fwd 5′-AGC​GCG​TCC​GGA​GCC​ACC​ATG​ACC​ACA​GAG​TCA​GAG​CCC​TTG​TTT​GGA​G-3′ 
VE-PTP 1,998 XhoI Rev 5′-GCC​GCC​TCT​AGA​CTA​ATG​TCT​CGA​GTA​GAT​TGC​ATC​TCT​GTG​ATA​C-3′ 
pAmCyan-VE-PTP 1–1,650 Integrated DNA Technologies VEPTP 1 BspEI Fwd 5′-AGC​GCG​TCC​GGA​GCC​ACC​ATG​CTG​AGG​CAT​GGA​GCC​CTA​ACG​GCC​TTG-3′ 
VEPTP 1,650 XhoI Rev 5′-GCC​GCC​CTC​GAG​GCT​AGC​TTT​CTG​TCT​GCA​GAT​GAA​GAA​GGC-3′ 
Plasmids   
mPlum-C1 Clontech 632527 
pAmCyan1-C1 Clontech 632441 
pNH-TrxT Addgene 26106 
VE-cad-Dendra2 Daneshjou et al., 2015  N/A 
VE-cadherin-GFP Shaw et al., 2001  N/A 
VE-cadherin tension FRET biosensor Conway et al., 2013  N/A 
pTriEx-Rac1 FLARE.dc biosensor WT MacNevin et al., 2016  N/A 
pTriEx-RhoA FLARE.sc biosensor WT Pertz et al., 2006  Addgene: 12150 
siRNA target sequences   
ON-TARGETplus ARHGEF2 siRNA Dharmacon J-009883-09; 5′-CCA​CGG​AAC​UGG​CAU​UAC​U-3′ 
ON-TARGETplus Human ARHGEF1 siRNA SMARTpool Dharmacon J-009421; 
1. 5′-UGA​CGU​GGC​GGG​UGA​CUA​A-3′, 
2. 5′-AAACYGGUGUGCUCUCAUC-3′, 
3. 5′-5′-CCA​CGG​CCC​UUC​GGA​AAG​U-3′, 
4. 5′-UAU​ACG​AGC​UGG​UGG​CAC​A-3′ 
ON-TARGETplus human ARHGEF18 siRNA SMARTpool Dharmacon J-009654; 
1. 5′-UCA​GGG​CGC​UUG​AAA​GAU​A-3′, 
2. 5′-GCA​GUG​ACC​GGA​AUU​AUG​U-3′, 
3. 5′-CAC​AAC​GCA​UAA​CCA​AAU​A-3′, 
4. 5′-GGA​CGC​AAC​UCG​GAC​CAA​U 
ON-TARGETplus human ARHGEF28 siRNA SMARTpool Dharmacon J-024506; 
1. 5′-CAA​GUA​AUC​UAC​AGU​CGA​A-3′, 
2. 5′-CAU​AAA​GAC​UUA​CGC​AAA​G-3′, 
3. 5′-GGC​UUG​AGA​UCC​UAA​AUA​A-3′, 
4. 5′-GAA​GAU​AUA​AAG​CGU​GUC​A-3′ 
ON-TARGETplus PTPRB siRNA Dharmacon J-004994-07; 5′-GAU​CUA​ACA​UUG​CGC​AAC​A-3′ 
siGENOME NT siRNA pool Dharmacon D-001206-13 
Reagents   
X-tremeGENE HP DNA transfection reagent Sigma Aldrich 6366244001 
Gene silencer transfection reagent Genlantis T500750 
Pierce Protein G Agarose ThermoFisher 20398 
Protein A/G PLUS-Agarose Santa Cruz sc-2003 
RhoA G17A Agarose beads Abcam ab211183 
Human α-thrombin Enzyme Research Laboratories HT 5142 
ROCK inhibitor III Santa Cruz sc-203237 
RhoA activator I Cytoskeleton, Inc. CN01 
QC colloidal Coomassie stain BioRad 1610803 
Cell lines and reagentsCompanyCatalog number and sequences
Cell lines   
HPAECs Lonza cc-2530 (lot numbers 329447, 447095, 466719, 598033, 662151, 4F3034) 
CHO-K1 ATCC TCC CCL-61 
Antibodies   
Phosphotyrosine antibody (PY20) Abcam ab16389 
Phosphotyrosine antibody, clone 4G10 EMD Millipore 05-321X 
VE-cadherin antibody Santa Cruz sc-6458 
Phospho-VE-cadherin (Tyr685) EMD Millipore ABT1760 
Phospho-VE-cadherin (Tyr658) polyclonal antibody ThermoFisher  44-1144G 
Phospho-VE-cadherin (Tyr685) polyclonal antibody Abcam ab119785 
Phospho-VE-cadherin (Tyr731) Polyclonal antibody ThermoFisher  44-1145G 
TIE2 antibody Abcam ab58302 
human/mouse phospho-Tie-2 (Y992) antibody R&D Systems AF2720 
Cingulin polyclonal antibody Novus Biologicals NBP1-89602 
GEF-H1 antibody Abcam ab155785 
Anti–GEF-H1 antibody [B4/7] Abcam ab90783 
p114RhoGEF (ARHGEF18) monoclonal antibody Novus Biologicals NBP2-43546 
p115RhoGEF (D-11; ARHGEF1) monoclonal antibody Santa Cruz sc-166341 
RGNEF (ARHGEF28) polyclonal antibody Abcam ab157095 
GST antibody (A-6) Santa Cruz sc-374171 
6x-His Tag monoclonal antibody ThermoFisher 4E3D10H2/E3 
GFP Tag monoclonal antibody ThermoFisher  GF28R 
PTPβ antibody (C-20) Santa Cruz sc-1114 
RPTPβ antibody BD Biosciences 610180 
Alexa Fluor 488 Phalloidin Invitrogen A12379 
Mouse monoclonal anti-β-tubulin Sigma Aldrich T8328 
Anti-mouse IgG (Fc) antibody Aviva Systems Biology OARD00003 
Primers   
pNH-TRXT-VE-PTP aa 1,651–1,998-6xHis Integrated DNA Technologies VE-PTP aa 1,651 EcorRI Fwd; 5′-ATA​GGA​GAA​TTC​GCC​ACC​ATG​AGG​GAA​AGG​CCA​TCT​GCC-3′ 
VE-PTP aa 1,998 HindIII Rev; 5′-AAA​CCT​AAG​CTT​ATG​TCT​CGA​GTA​GAT​TGC-3′ 
mPlum VE-PTP 1,422–1,998 D1871A Integrated DNA Technologies VE-PTP D1871A Fwd 5′-GCC​AGC​CCA​TGG​GGT​CCC​AGA​G-3′ 
VE-PTP D1871A Rev 5′-CCA​TGG​GCT​GGC​CAC​ACC​GTG​T-3′ 
mPlum VE-PTP 1,611–1,998 Integrated DNA Technologies VE-PTP 1,611 BspEI Fwd 5′-AGC​GCG​TCC​GGA​GCC​ACC​ATG​ACC​ACA​GAG​TCA​GAG​CCC​TTG​TTT​GGA​G-3′ 
VE-PTP 1,998 XhoI Rev 5′-GCC​GCC​TCT​AGA​CTA​ATG​TCT​CGA​GTA​GAT​TGC​ATC​TCT​GTG​ATA​C-3′ 
pAmCyan VE-PTP 1,651–1,998 Integrated DNA Technologies VE-PTP 1,651 BspEI Fwd 5′-GAC​ACG​TCC​GGA​GCC​ACC​ATG​AGG​GAA​AGG​CCA​TCT​GCC​CGG​CTC​AGC-3′ 
VE-PTP 1,998 XhoI Rev 5′-GGG​CCC​CTC​GAG​TTA​ATG​TCT​CGA​ATA​GAT​TGC​ATC​TCT​GTG​ATA​CTC-3′ 
mPlum VE-PTP 1–1,650 Integrated DNA Technologies VE-PTP 1 BspEI 5′ Fwd 5′-AGC​GCG​TCC​GGA​GCC​ACC​ATG​CTG​AGG​CAT​GGA​GCC​CTA​ACG​GCC​TTG-3′ 
VE-PTP 1,650 XhoI Rev 5′-GCC​GCC​CTC​GAG​GCT​AGC​TTT​CTG​TCT​GCA​GAT​GAA​GAA​GGC-3′ 
GST-GEFH1 572–876 Integrated DNA Technologies GEFH1 572 EcoRI Fwd 5′-GGA​CTC​GAA​TTC​GAG​CCA​CCA​TGA​CAT​GCC​CAT​CCA​GGG​AGG​AC-3′ 
GEFH1 876 HindIII Rev 5′-AGG​TCC​AAG​CTT​TTA​ATC​CAC​AGG​TCT​GCG​GGC​CCA-3′ 
GST-GEFH1 731–985 Integrated DNA Technologies GEFH1 731 EcoRI Fwd 5′-GGA​CTC​GAA​TTC​GAG​CCA​CCA​TGA​GAT​CAC​CGC​AAG​AGG​AGG​CG-3′ 
GEFH1 985 HindIII Rev 5′-AGG​TCC​AAG​CTT​TTA​GCT​CTC​GGA​GGC​TAC​AGC​CTC-3′ 
GST-GEFH1 731–876 Integrated DNA Technologies GEFH1 731 EcoRI Fwd 5′-GGA​CTC​GAA​TTC​GAG​CCA​CCA​TGA​GAT​CAC​CGC​AAG​AGG​AGG​CG-3′ 
GEFH1 876 HindIII REV 5′-AGG​TCC​AAG​CTT​TTA​ATC​CAC​AGG​TCT​GCG​GGC​CCA-3′ 
GST-GEFH1 731–925 Integrated DNA Technologies GEFH1 731 EcoRI Fwd 5′-GGA​CTC​GAA​TTC​GAG​CCA​CCA​TGA​GAT​CAC​CGC​AAG​AGG​AGG​CG-3′ 
GEFH1 925 HindIII Rev 5′-AGG​TCC​AAG​CTT​TTA​CCC​CAG​TTC​CTG​CCT​CTC​TCG-3′ 
pAmCyan-VE-PTP 1,611–1,998 Integrated DNA Technologies VE-PTP 1,611 BspEI Fwd 5′-AGC​GCG​TCC​GGA​GCC​ACC​ATG​ACC​ACA​GAG​TCA​GAG​CCC​TTG​TTT​GGA​G-3′ 
VE-PTP 1,998 XhoI Rev 5′-GCC​GCC​TCT​AGA​CTA​ATG​TCT​CGA​GTA​GAT​TGC​ATC​TCT​GTG​ATA​C-3′ 
pAmCyan-VE-PTP 1–1,650 Integrated DNA Technologies VEPTP 1 BspEI Fwd 5′-AGC​GCG​TCC​GGA​GCC​ACC​ATG​CTG​AGG​CAT​GGA​GCC​CTA​ACG​GCC​TTG-3′ 
VEPTP 1,650 XhoI Rev 5′-GCC​GCC​CTC​GAG​GCT​AGC​TTT​CTG​TCT​GCA​GAT​GAA​GAA​GGC-3′ 
Plasmids   
mPlum-C1 Clontech 632527 
pAmCyan1-C1 Clontech 632441 
pNH-TrxT Addgene 26106 
VE-cad-Dendra2 Daneshjou et al., 2015  N/A 
VE-cadherin-GFP Shaw et al., 2001  N/A 
VE-cadherin tension FRET biosensor Conway et al., 2013  N/A 
pTriEx-Rac1 FLARE.dc biosensor WT MacNevin et al., 2016  N/A 
pTriEx-RhoA FLARE.sc biosensor WT Pertz et al., 2006  Addgene: 12150 
siRNA target sequences   
ON-TARGETplus ARHGEF2 siRNA Dharmacon J-009883-09; 5′-CCA​CGG​AAC​UGG​CAU​UAC​U-3′ 
ON-TARGETplus Human ARHGEF1 siRNA SMARTpool Dharmacon J-009421; 
1. 5′-UGA​CGU​GGC​GGG​UGA​CUA​A-3′, 
2. 5′-AAACYGGUGUGCUCUCAUC-3′, 
3. 5′-5′-CCA​CGG​CCC​UUC​GGA​AAG​U-3′, 
4. 5′-UAU​ACG​AGC​UGG​UGG​CAC​A-3′ 
ON-TARGETplus human ARHGEF18 siRNA SMARTpool Dharmacon J-009654; 
1. 5′-UCA​GGG​CGC​UUG​AAA​GAU​A-3′, 
2. 5′-GCA​GUG​ACC​GGA​AUU​AUG​U-3′, 
3. 5′-CAC​AAC​GCA​UAA​CCA​AAU​A-3′, 
4. 5′-GGA​CGC​AAC​UCG​GAC​CAA​U 
ON-TARGETplus human ARHGEF28 siRNA SMARTpool Dharmacon J-024506; 
1. 5′-CAA​GUA​AUC​UAC​AGU​CGA​A-3′, 
2. 5′-CAU​AAA​GAC​UUA​CGC​AAA​G-3′, 
3. 5′-GGC​UUG​AGA​UCC​UAA​AUA​A-3′, 
4. 5′-GAA​GAU​AUA​AAG​CGU​GUC​A-3′ 
ON-TARGETplus PTPRB siRNA Dharmacon J-004994-07; 5′-GAU​CUA​ACA​UUG​CGC​AAC​A-3′ 
siGENOME NT siRNA pool Dharmacon D-001206-13 
Reagents   
X-tremeGENE HP DNA transfection reagent Sigma Aldrich 6366244001 
Gene silencer transfection reagent Genlantis T500750 
Pierce Protein G Agarose ThermoFisher 20398 
Protein A/G PLUS-Agarose Santa Cruz sc-2003 
RhoA G17A Agarose beads Abcam ab211183 
Human α-thrombin Enzyme Research Laboratories HT 5142 
ROCK inhibitor III Santa Cruz sc-203237 
RhoA activator I Cytoskeleton, Inc. CN01 
QC colloidal Coomassie stain BioRad 1610803 

Antibodies, reagents, primers, cell lines, siRNA, and vectors used in experiments throughout this study. Company, catalog numbers, and sequences (if applicable) are indicated. Fwd, forward; N/A, not applicable; Rev, reverse.

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