ZO-1 (D7D12) Rabbit mAb #8193

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	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

Cells were harvested and lysed in NP-40 buffer (Elpis Biotech, Inc., Daejeon, Korea) supplemented with a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). To address phosphorylation events, an additional set of phosphatase inhibitors (Cocktail II; Sigma-Aldrich; Merck KGaA) was added to the NP-40 buffer. Protein concentration was determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). The same volume of 2X Laemmli sample buffer (Elpis Biotech, Inc.) was added to each lysate, and each protein sample (10 µg/sample) was immediately boiled for 5 min at 100°C. Insoluble material was centrifuged at 14,000 × g at 25°C. Total cell lysates (5×106 cells/sample) were subjected to SDS-PAGE (10 µg/well) on a gel containing 15% (w/v) acrylamide under reducing conditions. Separated proteins were transferred to nitrocellulose membranes (EMD Millipore). The membranes were blocked with 5% skim milk and western blot analysis was performed. Chemiluminescence was detected using an enhanced chemiluminescence kit (Advansta, Inc., Menlo Park, CA, USA) and an Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK). Primary antibodies against the following proteins were incubated with membranes overnight at 4°C: E-cadherin (cat. no. 3195; 1:1,000), vimentin (cat. no. 5741; 1:1,000), tight junction protein 1 (ZO-1; cat. no. 8193; 1:1,000), EGFR (cat. no. 2232; 1:1,000), phospho-Smad2/3 (Ser465/467/Ser423/425; cat. no. 8828; 1:1,000), Smad2/3 (cat. no. 5678; 1:1,000), phospho-FAK (Tyr397; cat. no. 3283; 1:1,000), phospho-FAK (Tyr925; cat. no. 3284; 1:1,000), FAK (cat. no. 3285; 1:1,000), phospho-Syk (Tyr323; cat. no. 2715; 1:1,000), phospho-Syk (Tyr525/526; cat. no. 2710; 1:1,000), Syk (cat. no. 13,198; 1:1,000), phospho-Src (Tyr416; cat. no. 6943; 1:1,000), Src (cat. no. 2123; 1:1,000), extracellular signal-regulated kinase (ERK; cat. no. 9102; 1:1,000), phospho-ERK (cat. no. 9101; 1:1,000), phospho-p38 (cat. no. 9211; 1:1,000), p38 (cat. no. 9212; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9258; 1:1,000) phospho-JNK (cat. no. 4671; 1:1,000), and β-actin (cat. no. 4967; 1:1,000; Cell Signaling Technology, Inc.); and VEGF receptor 1 (VEGF-R1; cat. no. sc-271789; 1:500), VEGF-R2 (cat. no. sc-393163; 1:500), 12-LOX (cat. no. sc-365194; 1:500) and 15-LOX (cat. no. sc-133085; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); and α-smooth muscle actin (α-SMA; cat. no. bs-10196R; 1:500; BIOSS, Beijing, China) Following this, the membranes were incubated with the following secondary antibodies for 1 h at room temperature: Goat anti-mouse-horseradish peroxidase (HRP; cat. no. K0211589; 1:3,000) or goat anti-rabbit-HRP (cat. no. K0211708; 1:3,000) (both from Koma Biotech, Seoul, Korea).

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for ZO-1 (D7D12) Rabbit mAb #8193 below.

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