| Antibodies |
| Diap1(mDia1) Antibody |
Cell Signaling Technology |
Cat# 5486; RRID: AB_10828440 |
| FHOD1 Antibody |
ECM Biosciences |
Cat# FM3521; RRID: AB_2104508 |
| Integrin β1 Antibody,non-function perturbing, K20 |
Santa Cruz |
Cat# sc-18887; RRID: AB_627006 |
| Integrin β1 Antibody,blocking, 4B4 |
Beckman Coulter |
Cat# 6603113; RRID: AB_10638675 |
| Integrin β1 Antibody, activated, clone HUTS-4 |
Merck |
Cat# MAB2079Z; RRID: AB_2233964 |
| Transferin Antibody,OKT9 |
Purified from hybridoma cells procured from National Centre for Cell Science,Pune,India |
NA |
| FR Antibody, MOv 19 |
Dr Silvana Canevari (Fondazione IRCCS Istituto Nazionale dei tumori di Milano,Italy) |
NA |
| CD59 Antibody, MEM-43 |
Abcam |
Cat# ab9182; RRID: AB_307053 |
| Talin Antibody, Clone 8D4 |
Sigma Aldrich |
Cat# SAB4200694 |
| Vinculin Antibody, Clone SPM227 |
Abcam |
Cat# ab18058; RRID: AB_444215 |
| Paxillin Antibody |
BD Transduction Labs |
Cat# 610051; RRID: AB_397463 |
| AffiniPure Donkey Anti-Mouse IgG (H+L) |
Jackson ImmunoResearch Laboratories, Inc. |
Cat# 715-005-150; RRID: AB_2340758 |
| β-actin Antibody |
Sigma Aldrich |
Cat# A5060; RRID: AB_476738 |
| Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) |
Jackson ImmunoResearch Laboratories, Inc. |
Cat# 711-035-152; RRID: AB_10015282 |
| Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L) |
Jackson ImmunoResearch Laboratories, Inc. |
Cat# 115-035-003; RRID: AB_10015289 |
| CaptureSelect Biotin Anti-IgG-Fc (Multi-species) Conjugate |
Thermo Scientific |
Cat# 7102852100 |
| Chemicals, Peptides, and Recombinant Proteins |
| Human Plasma Fibronectin Purified Protein |
Merck (Sigma Aldrich) |
Cat# FC010 |
| H-Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala-OH trifluoroacetate salt (Disulfide bond between Pen2 and Cys9) |
Bachem |
Cat# H-3964 |
| SFK Inhibitor; PP2 |
Calbiochem (Merck) |
Cat# 529573 |
| FAK Inhibitor; PF 573228 |
Sigma Aldrich |
Cat# PZ0117 |
| C3 exoenzyme; Rho Inhibitor I |
Cytoskeleton, Inc. |
Cat# CT04 |
| Rho Activator II |
Cytoskeleton, Inc. |
Cat# CN03 |
| Formin Inhibitor; SMIFH2 |
Sigma Aldrich |
Cat# S4826 |
| Arp2/3 Inhibitor; CK666 |
Sigma Aldrich |
Cat# SML0006 |
| ROCK Inhibitor; Y-27632 dihydrochloride |
Sigma Aldrich |
Cat# Y0503 |
| MLCK Inhibitor; ML-7 |
Sigma Aldrich |
Cat# I2764 |
| 18:1 (Δ9-Cis) PC (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine |
Avanti Polar Lipids, Inc. |
Cat# 850375 |
| 16:0 Biotinyl Cap PE 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) |
Avanti Polar Lipids, Inc. |
Cat# 870277 |
| NeutrAvidin Protein, DyLight 650 |
Invitrogen (Thermo Fisher Scientific) |
Cat# 84607 |
| Avidin, NeutrAvidin Biotin-binding Protein |
Invitrogen (Thermo Fisher Scientific) |
Cat# A2666 |
| Cyclo[Arg-Gly-Asp-D-Phe-Lys(Biotin-PEG-PEG)] |
Peptides International |
Cat# PCI-3697-PI |
| Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane |
Sigma Aldrich |
Cat# L2020 |
| Collagen I Rat Protein, Tail |
GIBCO (Thermo Fisher Scientific) |
Cat# A1048301 |
| Poly-L-lysine solution, 0.01% |
Sigma Aldrich |
Cat# P4707 |
| Vitronectin from Human Plasma |
Sigma Aldrich |
Cat# V9881 |
| Methyl-β-cyclodextrin |
Sigma Aldrich |
Cat# C4555 |
| FuGENE® 6 Transfection Reagent, For plasmid DNA transfections |
Promega |
Cat# E2692 |
| HiPerFect Transfection Reagent, For siRNA transfections |
QIAGEN |
Cat# 301704 |
| Diaphanous (mDia)-related Formin Agonist, IMM01 |
Merck Millipore |
Cat# 509583 |
| 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine |
Sigma Aldrich |
Cat# 40227 |
| Filipin III from Streptomyces filipinensis
|
Sigma Aldrich |
Cat# F4767 |
| Alexa Fluor 488 phalloidin |
Invitrogen (Thermo Fisher Scientific) |
Cat# A12379 |
| Alexa Fluor 568 phalloidin |
Invitrogen (Thermo Fisher Scientific) |
Cat# A12380 |
| PLL(20)-g[3.5]-PEG(2)/PEG(3.4)biotin 50% |
SuSoS |
Cat# PLL(20)-g[3.5]-PEG(2)/PEG(3.4) biotin 50% |
| Proaerolysin |
Protein Technology Core (C-CAMP,Bangalore,India) |
N/A |
| Alexa 488- conjugated FLAER |
Protox Biotech |
Cat# FL1S |
| Hellmanex III |
Hellma Analystics (Sigma Aldrich) |
Cat# Z805939 |
| Experimental Models: Cell Lines |
| GG8;CHO-K1 cells (TRVb-1), devoid of transferrin receptor (TfR) were stably transfected with human TfR and EGFP-GPI |
Satyajit Mayor (Sabharanjak et al., 2002) |
N/A |
| U2OS; Human Osetosarcoma cells |
Clare Waterman (NHLBI,NIH,USA) (Case and Waterman, 2011) |
N/A |
| U2OS GG; U2OS cells stably transfected with GFP-GPI |
This paper |
N/A |
| Vin−/− MEFs;Mouse embryonic fibroblasts deficient in vinculin |
Daniel Rösel (Charles University in Prague, Czech Republic) (Janoštiak et al., 2014) |
N/A |
| GD3S-C37;3B2A cells (CHO cells stably expressing CD59 and DAF) stably expressing GD3 synthase |
Taroh Kinoshita (Osaka University, Japan)(Tashima et al., 2006) |
N/A |
| C84 DM2&3-C2;PGAP2/3 double mutant cell line stably expressing CD59 and DAF |
Taroh Kinoshita (Osaka University, Japan)(Maeda et al., 2007) |
N/A |
| C84 DM2&3-C2 + pMSCV-rPGAP2-zeo-hPGAP3;PGAP2/3 double mutant cells expressing PGAP2 and PGAP3 |
Taroh Kinoshita (Osaka University, Japan) (Maeda et al., 2007) |
N/A |
| Talin 1 deficient MEFs; Mouse embryonic fibroblasts deficient in talin 1 |
Mike Sheetz (Mechanobiology Institute, Singapore) (Zhang et al., 2008) |
N/A |
| CHOB2-α5-GFP;CHOB2 (that lack endogenous a5integrin) cells stably expressing GFP-a5 integrin |
Alan F Horwitz (University of Virginia,USA) (Laukaitis et al., 2001) |
N/A |
| FAK −/− MEFs; Mouse embryonic fibroblasts that have null mutations in FAK and p53 gene derived from E 8.0 embryo |
ATCC (llić et al., 1995) |
Cat# CRL-2644; RRID: CVCL_8954 |
| FAK +/+ MEFs;Mouse embryonic fibroblasts in which there is mutation in only the p53 gene and used as a control for the FAK−/− line |
ATCC (llić et al., 1995) |
Cat# CRL-2645; RRID: CVCL_8955 |
| MYG1;m-YFP GPI expressing CHO cells |
Satyajit Mayor (Suvrajit Saha PhD Thesis) |
N/A |
| Oligonucleotides |
| Primers used to generate the GFP-Vinculin Ld CA |
See STAR Methods
|
N/A |
| Primers used to generate the GFP-Vinculin Ld construct |
See STAR Methods
|
N/A |
| Primers used to generate the Lact C2 AAA-Ezrin AFBD-YFP construct |
See STAR Methods
|
N/A |
| ON-TARGETplus Non-targeting siRNA |
Dharmacon |
Cat# D-001810-01-05 |
| siGENOME Human FHOD1 siRNA SMART Pool |
Dharmacon |
Cat# M-013709-01-0005 |
| siGENOME Human DIAPH1 siRNA SMART Pool |
Dharmacon |
Cat# M-010347-02-0005 |
| Primers used to generate the mRuby2-GPI construct |
See STAR Methods
|
N/A |
| Recombinant DNA |
| Talin 2 shRNA |
Mike Sheetz (Mechanobiology Institute, Singapore) (Zhang et al., 2008) |
N/A |
| GFP-Vinculin WT;Full length |
Clare Waterman (NHLBI,NIH,USA) (Thievessen et al., 2013) |
N/A |
| mCherry-Vinculin WT;Full length |
Clare Waterman (NHLBI,NIH,USA) (Pasapera et al., 2010) |
N/A |
| GFP-Vinculin CA;constitutively active |
Clare Waterman (NHLBI,NIH,USA) (Case et al., 2015) |
N/A |
| GFP-Vinculin A50I;Talin non-binding |
Clare Waterman (NHLBI,NIH,USA) (Case et al., 2015) |
N/A |
| GFP-Vinculin A50I CA;Talin non-binding constitutively active |
Clare Waterman (NHLBI,NIH,USA) (Case et al., 2015) |
N/A |
| GFP-Vinculin Ld; Lipid non-binding |
This Paper |
N/A |
| GFP-Vinculin Ld CA; Lipid non-binding constiutively active |
This Paper |
N/A |
| GFP-Vinculin AB1; Actin non-binding |
Clare Waterman (NHLBI,NIH,USA) (Case et al., 2015) |
N/A |
| GFP-Vinculin AB1 CA; Actin non-binding constitutively active |
Clare Waterman (NHLBI,NIH,USA) (Case et al., 2015) |
N/A |
| GFP-Vinculin head; head domain of vinculin |
Clare Waterman (NHLBI,NIH,USA) (Case et al., 2015) |
N/A |
| Lact C2 Ezrin AFBD-YFP |
Protein Technology Core (C-CAMP,Bangalore,India) (Raghupathy et al., 2015) |
N/A |
| Lact C2-AAA Ezrin AFBD-YFP |
This Paper |
N/A |
| GFP-Vinculin AB2; Actin non-binding constitutively active |
Clare Waterman (NHLBI,NIH,USA) (Case et al., 2015) |
N/A |
| mRFP-tH |
John F.Hancock (University of Queensland,Australia) (Plowman et al.,2005) |
N/A |
| mRuby2-GPI |
This Paper |
N/A |
| pcDNA3-mRuby2 |
Michael Lin (Stanford University, USA) (Lam et al., 2012) |
Addgene plasmid # 40260; http://addgene.org/40260; RRID: Addgene_40260) |
| Software and Algorithms |
| MATLAB, Image processing toolbox |
Mathworks Inc. |
https://www.mathworks.com/products/matlab.html |
| Fiji Image analysis software |
Schindelin et al., 2012 |
https://fiji.sc |
| GraphPad Prism 7 software |
GraphPad |
https://www.graphpad.com/scientific-software/prism/ |
