Whole cell protein lysates were extracted using cell lysis buffer (Cell Signaling Technology) following the manufacturer’s instructions and protein concentration was determined using the Bradford Assay Kit (Sigma-Aldrich). Immunoblotting was performed according to standard methods. The following primary antibodies were used according to the manufacturer’s instructions: cortactin (#3502), phospho-cortactin Tyr421 (#4569), ERK1/2 (#4695), phoshpho-ERK1/2 Thr202/Tyr204 (#4370), GAPDH clone D16H11 (#5174), Src (#2109) and phospho-Src Tyr416 (#2101), gelsolin D9W8Y (#12953) (all rabbit host species and obtained from Cell Signaling Technology) and E-cadherin NCH-38 (#MA5-12547, mouse, Thermo Fisher Scientific). After Secondary HRP-labeled antibody (rabbit #1706515, mouse #1706516, both obtained from Bio-Rad Laboratories) incubation (1 h, RT) immunolabeling was detected using an enhanced Chemiluminescence Detection Kit (SignalFire ECL Reagent; Cell Signaling Technology) and the Molecular Imager ChemiDoc system (Image Lab Software; Bio-Rad Laboratories).
Gapdh clone d16h11
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
GAPDH (clone D16H11) is a primary antibody that recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a key enzyme involved in glycolysis, the metabolic pathway that converts glucose to energy. This antibody can be used to detect and quantify GAPDH expression in a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Tapi 0, by Merck Group (2 mentions)
Fibronectin, by Merck Group (2 mentions)
β actin ac 15, by Merck Group (2 mentions)
Ephrinb2, by R&D Systems (2 mentions)
Anti αsma clone 1a4, by Merck Group (2 mentions)
Ephrin a1, by R&D Systems (2 mentions)
Adam10, by Cell Signaling Technology (2 mentions)
Ephrin a2, by R&D Systems (2 mentions)
Ephrin a3, by R&D Systems (2 mentions)
Ephrin a4, by R&D Systems (2 mentions)
Ephrin b1, by R&D Systems (2 mentions)
Ephrin b3, by R&D Systems (2 mentions)
Hpa008999, by Merck Group (2 mentions)
Recombinant human and mouse tgf beta 1 protein, by R&D Systems (2 mentions)
Ephrin a5, by R&D Systems (2 mentions)
Ha tag c29f4, by Cell Signaling Technology (2 mentions)
Pdgf bb, by R&D Systems (2 mentions)
4 hydroxytamoxifen, by Merck Group (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
6 protocols using gapdh clone d16h11
1
Immunoblot Analysis of Cell Signaling
2
Western Blot Analysis of EMT Markers
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Protein was extracted from the cells using Pierce RIPA Buffer (Thermo Fisher Scientific, Rockford, IL, USA) with the cOmplete, EDTA‐free Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). A total of 20 μg whole cell extracts was loaded on mini protean TGX 4–15% gels (Bio‐Rad, Hercules, CA, USA) and transferred using the Trans‐Blot Turbo Blotting System (Bio‐Rad). The membranes were probed with the following primary antibodies: mAbs for E‐cadherin (clone 24E10; Cell Signaling Technology, Beverly, MA, USA), vimentin (clone D21H3; Cell Signaling Technology), Snail (clone C15D3; Cell Signaling Technology), SERPINI1 (clone 1D10; Sigma‐Aldrich), CHST11 (clone 1H3; Sigma‐Aldrich), or GAPDH (clone D16H11; Cell Signaling Technology) as a control at 4°C overnight. The secondary antibodies were peroxidase‐coupled goat anti‐rabbit or anti‐mouse antibodies and detected with Clarity Western ECL Substrate (Bio‐Rad), and the protein bands were visualized using the ImageQuant LAS 4000 mini system (GE Healthcare Life Sciences).
3
Ephrin-B2 Mutant Expression Analysis
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ephrin-B2 plasmids including HA-tagged full-length ephrin-B2wildtype, ephrin-B2ΔEcto-HA, ephrin-B2ΔJuxta-HA, ephrin-B2Δ182-194-HA and ephrin-B2Δ197-218-HA were kindly shared by Ira O. Daar (National Cancer Institute, National Institutes of Health, USA). Mouse and human recombinant PDGF-BB, ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5, ephrin-B1, ephrin-B2 and ephrin-B3 were purchased from R&D Systems, 18:1 LPA from Avanti Polar Lipids. Antibodies used for Western blotting include: mouse polyclonal anti-α-SMA (clone 1A4; Sigma-Aldrich), rabbit polycloncal collagen type I (ab34710, Abcam), rabbit monoclonal GAPDH (clone D16H11, Cell Signaling), mouse monoclonal β-actin (AC-15, Sigma), ephrin-B2 (P-20 Santa Cruz and HPA008999 Sigma), ADAM10 (#14194, Cell Signaling) and rabbit monoclonal HA-Tag (C29F4, Cell Signaling). Pharmacological inhibitors including BB-94 (Batimastat), GI254023X and TAPI-0 as well as phorbol 12-myristate 13-acetate (PMA), 4-hydroxytamoxifen, and fibronectin were purchased from Sigma-Aldrich. Recombinant human and mouse TGF-beta 1 protein was purchased from R&D.
4
Ephrin-B2 Mutant Expression Analysis
5
Signaling Assays in Cytotoxic T Cells
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In total, 0.2–1 × 106 CTLs were lysed using cold cell lysis buffer containing 50 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, and 0.25% sodium deoxycholate. Suppression of talin 1 was confirmed using an anti-talin 1 antibody (clone 8D4, Abcam). Talin head domain GFP fusion was detected using a polyclonal antibody against GFP (Invitrogen). Actin (clone AC-15, Sigma) or GAPDH (clone D16H11, Cell Signaling Technology) served as loading controls. For signaling assays, serum and IL-2 starved OT-1 CTLs were incubated with streptavidin polystyrene beads (Spherotech) coated with H-2Kb-OVA and ICAM-1 at a 1:1 ratio for various times at 37 °C and immediately lysed in 2× cold lysis buffer containing phosphatase inhibitors (1 mM NaF and 0.1 mM Na3VO4) and protease inhibitors (cOmplete mini cocktail, EDTA-free, Roche). Activation of PI3K and MAP kinase signaling was assessed by immunoblot for pAkt (Phospho-Akt (Ser473) Ab; Cell Signaling Technology) and pErk1/2 (Phospho-Thr202/ Tyr204; clone D13.14.4E; Cell Signaling Technology). Additional information about antibodies may be found in Supplementary Table 1 . Uncropped images of blots are provided in Supplementary Fig. 12 .
6
Western Blotting Protein Analysis
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Selected cell lines were maintained in 10-cm culture dishes and analyzed when 70%-80% confluent. Western blot analysis was performed as described previously (16) . Primary antibodies were specific for B2M (clone EP2978Y, ref 75853, Abcam, Cambridge, UK) and GAPDH (clone D16H11, Cell Signaling Technology, Danvers, MA, Cat #5174). Immunoreactivity was analyzed with the ECL-Plus Kit (Amersham Biosciences Co., Buckinghamshire, UK) using the ChemiDoc MP System (Bio-Rad Laboratories, Hercules, CA).
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