TC10 (Novus, Littleton, CO, USA; 07-2151; rabbit polyclonal used at 1:500 for western blots), p190 (BD Transduction Laboratories; 610149; Clone 30/p190; mouse monoclonal), p120 (Abcam, Cambridge, UK; ab2922; Clone B4F8; mouse monoclonal), Exo70 (Santa Cruz Biotechnology; sc-365825; Clone D-6; mouse monoclonal), Vamp7 (Abcam; ab36195; Clone 158.2; mouse monoclonal), MT1-MMP-Hinge region (Millipore, Burlington, MA, USA; AB6004; rabbit polyclonal), MT1-MMP (Millipore; MAB3328; Clone LEM-2/15.8; mouse monoclonal), MT1-MMP (Abcam; ab38971; rabbit polyclonal), Cortactin (Abcam; ab3333; Clone 0.T.21; mouse monoclonal, used at 1:600), Cortactin (Abcam; ab81208; Clone EP1922Y; rabbit monoclonal), Cortactin (Santa Cruz Biotechnology; sc-30771; G-18; goat polyclonal), MYC (Cell Signaling Technology, Danvers, MA, USA; mab2278; Clone 71D10; rabbit monoclonal), FLAG (Sigma; F1804; Clone M2; mouse monoclonal), EGFP (Roche; 11814460001; Clones 7.1 and 13.1; mixture mouse monoclonal), Rac1 (Millipore; 05-389; Clone 23A8; mouse monoclonal), Cdc42 (Santa Cruz Biotechnology; sc-8401; Clone B-8; mouse monoclonal), and RhoA (Santa Cruz Biotechnology; sc-418; Clone 26C4; mouse monoclonal). Unless otherwise stated, all primary antibodies were used at 1:200 dilution for immunofluorescence and 1:1000 for western blotting. Antibody specificities for TC10 vs Cdc42 are shown in Supplementary Figure 19z .
Cortactin
Manufactured by Abcam
Sourced in United States, United Kingdom
Cortactin is a protein that plays a role in the organization of the actin cytoskeleton. It is involved in the regulation of actin-related processes such as cell migration and endocytosis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Xp rabbit mab, by Cell Signaling Technology (2 mentions)
Pmcherry c1, by Takara Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Protein kinase buffer, by New England Biolabs (2 mentions)
Cofilin, by Abcam (2 mentions)
Adp glo kinase assay, by Promega (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Cortactin, by Santa Cruz Biotechnology (1 mentions)
Mt1 mmp, by Abcam (1 mentions)
Vamp7, by Abcam (1 mentions)
Mt1 mmp, by Merck Group (1 mentions)
Cdc42, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Versadoc mp 4000 system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Dapi staining, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
16 protocols using cortactin
1
Antibody Panel for Cell Signaling
2
Optogenetic Control of Cofilin Dynamics
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Antibodies were from the following sources: Cofilin (D3F9) XP® Rabbit mAb (Cell Signaling #5175), β-Actin (8H10D10) Mouse mAb (Cell Signaling #3700), Tks5 (Santa Cruz Biotechnology; sc-30122), Cortactin (Abcam; ab33333). The cDNA of the LOV2 domain from Avena sativa (oat) Phototropin1 (L404-L547) was used to generate photo-sensitive constructs. Three variants of LOV2 were used: wild-type, dark state mutant (C450A, L514K, G528A, L531E, and N538E), and lit state mutant (I510E/I539E) (Supplementary Table 2 ). 24 (link) The cDNA of full-length rat cofilin was used for all constructs. The Z affibodies that selectively bind dark state LOV2 have been described elsewhere. 24 (link) For transient expression in mammalian cells, constructs were cloned into pmCherry-C1 (Clontech Laboratories, Inc.). Cells were transfected with Lipofectamine 2000 (Life Technologies) using the manufacturer’s protocol 24 h before imaging. For imaging of living cells, cells were co-transfected with mCherry Z-lock cofilin and a membrane-anchored yPet (KRas C-terminus) to visualize the cell edge. 51
3
Phosphorylation of SYK, Cortactin, and Cofilin
4
Protein Extraction and Western Blot Analysis
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Cells were scraped in cold PBS and centrifuged down (500 × g) to remove methanol. The pellet was resuspended in cold RIPA buffer (Pierce), supplemented with Complete protease inhibitor cocktail tablets (Roche). After centrifugation at 13,000 × g for 10 min, the supernatant was collected for further analysis. For protein extraction from mouse tumour tissues, frozen tissues were ground in a mortar and pestle and then immersed in cold RIPA buffer plus protease inhibitor. Further homogenisation was performed by passing the tissues 5–10 times through a 21-gauge needle. After centrifugation at 13,000 × g for 10 min, the supernatant was collected and mixed with 1X SDS sample buffer. Protein samples were loaded onto 7 % or 12 % SDS-PAGE gels, running in Mini Trans-Bolt module (Bio-Rad). After gel electrophoresis, proteins were transferred to PVDF membranes (Millipore). The membranes were incubated with primary antibodies against cortactin (1:2000, Abcam) and α-Tubulin loading control (1:5000, Abcam) overnight at 4 °C after a 45 min blocking. Horseradish peroxidise-conjugated goat anti-mouse and anti-rabbit (1:10000, Bio-Rad) secondary antibodies were applied afterwards. SuperSignal chemiluminescent substrate (Pierce) was added to the membranes which were visualised using a VersaDoc MP4000 system (Bio-Rad).
5
Immunofluorescence Staining of Cortactin
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Sterile coverslips were placed in a 24-well plate and cancer cells were seeded at a density of 1.5 × 105 cells per well. After overnight incubation at 37 °C, cells were adherent to the coverslips. Treated cells were washed in ice cold PBS and fixed in 100 % methanol at −20 °C for 10 min, followed by washing in ice cold PBS twice, shaking gently. Usage of 0.2 % Triton X-100 (Sigma) in PBS to permeabilise samples was for no more than 10 min, followed by 3 times of wash in PBST. Cells were then blocked in block buffer (3 % normal goat serum and 0.5 % BSA in 0.01 M PBS) for 30 min and sequentially incubated with primary antibody cortactin (1:300, Abcam) overnight at 4 °C. The goat anti-rabbit Texas Red (1:1500, Abcam) secondary antibody was applied in dark for 1 h, followed a counter DAPI staining (Molecular Probes). All the coverslips were sealed onto microscope slides using ProLong Gold antifade reagent (Molecular Probes) and kept in dark for 24 h. Fluorescence images were visualised using confocal microscope FV1000 (Olympus).
6
Western Blot Analysis of RPE Proteins
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Lysates were prepared from RPE-choroid preparations from freshly dissected mice (Cryba1fl/fl, Cryba1 cKO), rat (WT and Nuc1), normal, and AMD RPE cells from human tissues, fetal human RPE cell cultures, and OCM3 cell line. Total protein (50 μg) was loaded for fetal human RPE cell culture and 10 μg protein was loaded for the other samples. Western blots were performed as described previously.12 (link),13 (link) The primary antibodies used in this study were βA3/A1-crystallin (Cat# sc-22398; Santa Cruz Biotechnology and Cat# ab151722; Abcam, Cambridge, MA, USA) as well as a polyclonal antibody developed in our laboratory and previously characterized,7 (link) E-cadherin (Cat# 610181, BD Biosciences, Franklin Lakes, NJ, USA), Vimentin (Cat# 550513; BD Biosciences and Cat# MA5-11883; Thermo Fisher Scientific, Waltham, MA, USA), β-Catenin (Cat# ab16051; Abcam), CortActin (Cat# ab33333; Abcam), phospho(Y421)-CortActin (Cat# 4569S; Cell Signaling Technology, Danvers, MA, USA), Snail1 (Cat# 14-9859-82; Invitrogen, Carlsbad, CA, USA), and Actin (Cat# A2066; Sigma Aldrich Corp., St. Louis, MO, USA). Secondary antibodies were peroxidase labeled goat anti-rabbit (Cat# 074-1506; KPL, Gaithersburg, MD, USA) and peroxidase labeled goat anti-mouse (Cat# 074-1806; KPL).
7
Comprehensive Tissue Analysis Protocol
8
Immunoprecipitation and Western Blot Analysis
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Lysate samples containing 200–600 μg cell were precleared using 25 μl protein G magnetic beads (New England BioLabs, Frankfurt, Germany); after 1‐h incubation and removal of the beads, the precleared lysate was incubated with 3 μg mAb33 anti‐KV10.1 antibody for 1 h, and 25 μl clean protein G magnetic beads were added and incubated for 1 h. The recovered beads were then washed with 50 mM Tris–HCl pH 7.4, 300 mM NaCl, 5 mM EDTA, and 0.1% Triton X‐100. Bound proteins were eluted with PAGE loading buffer. Samples were separated in 3–8% or 4–12% gradient polyacrylamide precast gradient gels (Thermo Fisher Scientific), transferred to nitrocellulose membranes, and immunoblotted with polyclonal anti‐KV10.1 antibody (9391).
For Western blot of additional proteins, the following antibodies were used: pan‐14‐3‐3 (sc‐629, Santa Cruz Biotechnology, Santa Cruz, CA), Actin (I19, Santa Cruz), phospho‐Aurora A (#3079, Cell Signaling Technologies, Danvers, MA), Aurora A (#12100; Cell Signaling), Calnexin (ADI SPA 860, Enzo Life Sciences, Lörrach, Germany), CortActin (ab81208, Abcam), phospho‐cortActin (Y466, SAB4504373, and Y421, SAB4504372, both from Sigma), γ‐tubulin (sc7396, Santa Cruz), and human transferrin receptor (612125, BD).
For Western blot of additional proteins, the following antibodies were used: pan‐14‐3‐3 (sc‐629, Santa Cruz Biotechnology, Santa Cruz, CA), Actin (I19, Santa Cruz), phospho‐Aurora A (#3079, Cell Signaling Technologies, Danvers, MA), Aurora A (#12100; Cell Signaling), Calnexin (ADI SPA 860, Enzo Life Sciences, Lörrach, Germany), CortActin (ab81208, Abcam), phospho‐cortActin (Y466, SAB4504373, and Y421, SAB4504372, both from Sigma), γ‐tubulin (sc7396, Santa Cruz), and human transferrin receptor (612125, BD).
9
Western Blot Analysis of Myosin X and Cortactin
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Total protein was extracted from tissues and measured with a BCA protein assay kit (Bioswamp, Wuhan, China). Samples containing 20 μg of proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and transferred to polyvinyl difluoride membranes (Guge Biotechnology, Wuhan, China). After blocking with 5% fat-free skim milk for 1 h at room temperature, the membranes were incubated overnight with primary antibodies against Myosin X (1:100, Abcam, Cambridge, UK) and Cortactin (1:100, Abcam). After washing three times in TBS-T buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were then processed using an ECL chemiluminescence reaction kit and followed by exposure on chemiluminescent film to visualize the proteins. GAPDH quantification was used as an internal standard to correct for variations in total protein loading. Densitometric analysis of the blots was performed using the software TANON GIS.
10
Protein Extraction and Western Blot Analysis
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Cells were harvested using the total protein extraction kit (KeyGEN BioTECH, Nanjing, China). Samples were incubated for 0.5 h on ice with agitation and then centrifuged at 14 000×g for 15 min. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Pierce). Protein samples were subjected to electrophoresis on SDS-polyacrylamide gradient gels, transferred to a PVDF membrane, and blocked in 5% non-fat milk in TBST (phosphorylated proteins were blocked in 5% BSA in TBST) for 2 h at room temperature. Blots were incubated with primary antibodies to the following proteins: ezrin (Abcam), cortactin (Abcam), E-cadherin (CST), α-SMA (Abcam), Slug (CST), Snail (Abcam), Twist (Abcam), Twist2 (Abcam), phosphorylated ezrin at Y-567 (CST), and GAPDH (Beyotime). GAPDH was used on the same membrane as a loading control. The signal was detected after incubation with anti-rabbit or anti-mouse IgG secondary antibody (Bioworld) coupled to peroxidase, using ECL (Millipore). Protein expression levels were evaluated by densitometric analysis.
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