Cells were washed twice with PBS and total cellular proteins were extracted using a radioimmunoprecipitation assay (0.5% Nonidet P-40, 10 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1% sodium dodecyl sulfate). Whole cell lysates were sonicated, boiled at 95°C for 5 min and then chilled on ice for 10 min. The relative level of protein expression was determined using specific antibodies. Antibodies used for Western blots were: rabbit antibodies against Syntaxin 2 (ab12369, Abcam), α-SMA (Sigma), phospho-FAK (Tyr-397, Biosource International, Camarillo, CA), ERK 1/2, phospho-ERK 1/2 (Thr202/Tyr204), MMP-3 and TIMP-1(Cell Signaling Technology), GAPDH (ab70699, Abcam); and mouse monoclonal antibodies against FAK (Upstate Biotechnology, NY). The immunoreactions were visualized using an enhanced ECL detection kit (Amersham Pharmacia Biotech), exposed to X-ray film and quantified using a video documentation system (Gel Doc 2000, Bio-Rad).
Ab12369
Manufactured by Abcam
Sourced in China
Ab12369 is a lab equipment product manufactured by Abcam. It is designed for use in scientific research applications. The core function of this product is to provide a specific tool or instrument for laboratory work, but further details on its intended use or capabilities are not available in this concise, unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Ab70699, by Abcam (1 mentions)
Enhanced ecl detection kit, by Cytiva (1 mentions)
α sma, by Merck Group (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Gel doc 2000, by Bio-Rad (1 mentions)
Dm4000b microscope, by Leica (1 mentions)
Ab86714, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by RiboBio (1 mentions)
As001, by ABclonal (1 mentions)
Chemiluminescence detection system, by Bio-Rad (1 mentions)
Minute plasma membrane protein isolation kit, by Invent Biotechnologies (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
4 protocols using ab12369
1
Western Blot Analysis of Cellular Proteins
2
Placental SXT2 Immunohistochemical Analysis
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Immunohistochemical analysis of placental tissues was performed on 4-μm paraffin sections. After dewaxing and rehydration, sections were treated with citric acid for antigen retrieval and blocked in 10% (wt/vol) Bovine Serum Albumin (BSA). Incubation with the primary antibody (polyclonal anti-SXT2 (ab12369, Abcam, Cambridge, MA; 1:500) was performed overnight at 4°C in a humid chamber. Slides were then washed with PBS and incubated with a secondary antibody (1:500; biotinylated secondary antibody; ZSGB-BIO) for 20 min at room temperature. Slides were then washed again in PBS and chromogenic detection was performed using a DAB (diaminobenzidine tetrahydro chloride) reagent kit was for 2–5 min, with hematoxylin used as counterstain. Imaging was performed with a Leica DM4000B microscope (Germany). Protein expression levels was performed with Image-Pro Plus 5.1 soft by two independent investigators blinded to the subgroups.
3
Immunofluorescence Assay for STX2 and PI3K
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STX2-specific shRNA treated trophoblasts (HTR-8 or primary human trophoblast cells) were cultured for 24 h. To permeabilize cells for immunofluorescence assays, incubation with 0.5% (vol/vol) TritonX-100 for 15 min at room temperature was performed. Cells were then blocked with 6% (wt/vol) goat serum, and incubated with anti- PI3K p85 (ab86714, Abcam; 1:100) overnight at 4°C. Cells were then washed and incubated with a fluorescence-labeled second antibody (anti-488; AS001, abclonal; 1:300) for 1 h at room temperature. Then, cells were incubated with the anti-SXT2 antibody (ab12369, Abcam; 1:100) for 1.5 h and with another fluorescence-labeled second antibody (anti-CY3; AS007, abclonal; 1:300) for 1 h. Cells were washed once again and stained with DAPI (Guangzhou RiboBio, China; 1 μg/mL) for 5 min at room temperature. Imaging was performed on a confocal fluorescence microscope.
4
Membrane Protein Isolation and Western Blot Analysis
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Cell lysis was conducted on ice with RIPA buffer (Sigma-Aldrich, St. Louis, United States). Cell lysates were centrifuged at 12,000 × g for 20 min and treated with 5x protein loading buffer. The membrane protein was isolated using the MinuteTM Plasma Membrane Protein Isolation Kit (SM-005, Invent Biotechnologies). Protein aggregates were then separated by SDS-PAGE and electro-transferred onto a PVDF membrane (Bio-Rad, Hercules, United States). Membranes were blocked in 5% (wt/vol) instant skim milk for 1 h at room temperature. Membranes were incubated at 4°C overnight with primary antibodies (SXT2 (ab12369, Abcam, Cambridge, MA; 1:1,000), p-AKT (ser473, #4060, CST; 1:1,000), AKT (#4685, CST; 1:1,000), p-GSK3β (ser9, #5558, CST; 1:1,000), β-catenin (#8480, CST; 1:1,000), β-Tubulin (#15115, CST; 1:5,000), PI3K p85 (#4257, CST; 1:1,000), PI3K p110α (#4249, CST; 1:1,000), PI3K p110β (#3011, CST; 1:1,000).
Following incubation with the primary antibody, membranes were then washed and incubated with secondary antibodies (1: 1,000; CST, Danvers, United States). Protein-antibody complexes were detected and quantified with a chemiluminescence detection system (Bio-Rad, United States?).
Following incubation with the primary antibody, membranes were then washed and incubated with secondary antibodies (1: 1,000; CST, Danvers, United States). Protein-antibody complexes were detected and quantified with a chemiluminescence detection system (Bio-Rad, United States?).
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