The tissue and cell samples were lysed in RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Jiangsu KeyGEN BioTECH, Nanjing, China) and the final concentration of lysates was adjusted to 1.5 μg/μl. When running western blots, the sample volume of each well was 10 μl, that is, 15 μg of total protein (15‐well electrophoresis comb, 1.5 mm). Syk (ab40781, Abcam) and p‐Syk (ab58575, Abcam) antibodies for human tissues and Syk (ab40781, Abcam), p‐Syk (ab58575, Abcam), MLCK (ab76092, Abcam), MLC (ab92721, Abcam), p‐MLC (ab2480, Abcam), ZO‐1 (ab96587, Abcam), and Occludin (ab216327, Abcam) antibodies for mouse tissues were used. The protein bands were visualized by a ChemiDoc Touch Imaging System (Bio‐Rad, USA) and quantified by the Image Lab software. Results were normalized to the GAPDH internal control.
Ab76092
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab76092 is a laboratory reagent. It is a product designed for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab216327, by Abcam (3 mentions)
Ab97023, by Abcam (2 mentions)
Ab53154, by Abcam (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ab58575, by Abcam (1 mentions)
Ab92721, by Abcam (1 mentions)
Ab40781, by Abcam (1 mentions)
Ab2480, by Abcam (1 mentions)
Ab96587, by Abcam (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Ab203567, by Abcam (1 mentions)
Total protein extraction kit, by Keygen Biotech (1 mentions)
Multispectral imaging system, by Analytik Jena (1 mentions)
Ab59348, by Abcam (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Image pro plus v6, by Media Cybernetics (1 mentions)
Enhanced chemiluminescence western blot detection reagents, by Advansta (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
P mlc, by Abcam (1 mentions)
9 protocols using ab76092
1
Western Blot Analysis of Signaling Proteins
2
Protein Expression Analysis in HPAF-II Cells
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A radioimmunoprecipitation assay was utilized to extract total proteins from the HPAF-II cells. The concentrations of the extracted proteins were measured by a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Fifty micrograms of proteins for each sample were loaded into each lane of 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, separated by gel electrophoresis, and transferred to polyvinylidene fluoride membranes. After blocking, blots were incubated with primary antibodies, including NF-κB p65 mAb (8242, CST, Boston, Mass), phospho-NF-κB p65 mAb (3033, CST), anti-MLCK antibody (Ab76092; Abcam, Cambridge, UK), and anti-TRIC antibody (Ab203567; Abcam) overnight at room temperature. After that, incubation was carried out with horseradish peroxidase-conjugated secondary antibodies (ab97023; Abcam) at room temperature for 1 hour and then detected by ECL reagents (BioRad, Shanghai, China) using X-ray film. Band density was measured by ImageJ (version 1.8; National Institutes of Health, Bethesda, Md). Protein expression of MLCK was detected by WES (ProteinSimple, San Francisco, Calif), using the Wes 66 to 440 kDa Mouse Master kit (PS-ST03-8; ProteinSimple) and Wes 12 to 230 kDa Master kit (SM-W002; ProteinSimple), according to the manufacturer's instructions.
3
Immunofluorescence Protocol for MLCK Detection
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The slides were prepared as described above for immunofluorescence. After blocking, the slides were treated with 3% hydrogen peroxide at room temperature for 10 minutes. Anti-MLCK antibodies (ab76092; Abcam) were added and incubated overnight. Then, cells were incubated with HRP-conjugated secondary antibodies (ab97023; Abcam) at room temperature for 1 hour. Slides were dyed with diaminobenzidine and counterstained with hematoxylin afterward. The slides were visualized using a fluorescence microscope (CX71; Olympus Corporation, Tokyo, Japan) at 400× magnification.
4
Analyzing Intestinal Protein Markers
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Colonic segments were isolated from mice and were then immediately stored in liquid nitrogen. Total protein was isolated from full thickness of intestinal wall using total protein extraction kit (Key GEN Bio TECH, Dalian, China). The blots on nitrocellulose filter membrane were probed with corresponding antibodies according to the manufacturer's instructions, respectively, at 4°C with gentle shaking, overnight. Antibodies to Bcl-2 (ab59348), Bax (ab53154), MLCK (ab76092), and occludin (ab216327) were purchased from Abcam. The bands were detected and quantified using multi-spectral imaging system (UVP, Cambridge, UK). The western blotting images were quantified by Image Lab (version 4.0.1; Bio-Rad, CA, USA).
5
Immunoblotting Analysis of Hippocampal Proteins
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Total proteins were extracted from differentially treated hippocampal neuronal cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) on ice. The protein concentration was determined with BCA Protein Assay kit (Beyotime Institute of Biotechnology). The proteins (50 µg) were isolated by 12% SDS-PAGE at room temperature. Following electrophoresis, the proteins were transferred onto polyvinylidene membranes (Thermo Fisher Scientific, Inc.) for 1.5 h. The membranes were incubated with 5% non-fat milk in TBS for 1.5 h at room temperature, followed by incubation with primary antibodies against MLCK (1:5,000; ab76092; Abcam), p-MLC (1:1,000; cat. no. 3675; Cell Signaling Technology, Inc.) and β-actin (1:2,000; BS6007MH; Biogot Technology Co., Ltd., Nanjing, China) overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:2,000; Bioworld Technology, China) for 2 h at room temperature. Bands were visualized with enhanced chemiluminescence western blot detection reagents (Advansta, Inc., Menlo Park, CA, USA) and analyzed using the Image-Pro Plus V6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
6
Evaluating MLC Protein Changes in DCs
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The supernatant of tumor cells was incubated with DCs as previously described. Changes in MLC protein levels in DCs in response to different treatments was determined using western blotting analysis. The antibodies used were anti-MLC (Abcam, ab76092, RRID: AB_1524000), anti-pMLC (Abcam, ab157747), and anti-β-actin (Abcam, ab8226, RRID: AB_306371). The dilution ratio of pMLC and MLC antibodies was 1/1000. Total cellular protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies overnight, followed by the corresponding secondary antibodies for 1 h at room temperature. β-Actin was used as a loading control. The signals were detected using an ECL western blotting detection kit (Abcam, ab133408).
7
Antibody Evaluation in Intestinal Cells
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TMEM16A antibodies (ab53213), MLCK antibodies (ab76092), cleaved caspase3 antibodies (ab2302), Bcl-2 antibodies (ab59348), and Bax antibodies (ab53154) were bought from Abcam (Hong Kong) Ltd. (Hong Kong, China). The TMEMD16A antibodies (14476S), phosphorylated ERK1/2 antibodies (#4370) and ERK1/2 antibodies (#4695), were bought from Cell Signaling (Boston, USA). The TMEMD16A antibodies (12652-I-AP) were bought from Proteintech Group (Chicago, USA). The rat intestinal epithelial cell line IEC-6 cells were bought from cell bank of Shanghai Institute (Shanghai, China). BrdU kit (ab126556) was obtained from Abcam (Hong Kong) Ltd. (Hong Kong, China). The cells used in this study were evaluated before experiment. Unless otherwise indicated, chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
8
Analyzing Hmgxb4 Knockout Mouse Tissues
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Brain or aortic tissues were harvested from 4‐month‐old WT and their littermate Hmgxb4 homozygous mice as we previously described (He et al., 2021 (link); Wen et al., 2017 , 2019 (link)). Tissues were ground with a glass homogenizer in 100 μL RIPA buffer (ThermoFisher Scientific, Cat. No. 89900) with 1% proteinase (ThermoFisher Scientific, Cat. No. A32963) and phosphatase inhibitor cocktail (ThermoFisher Scientific, Cat. No. A32957). After sonication and centrifugation of the lysate, proteins were quantified by BCA assay (ThermoFisher Scientific, Cat. No. 23225) and then loaded in a 9% SDS‐PAGE gel at 10–20 μg per lane. Antibodies used in this study are: HMGXB4 (Sigma, HPA000725, rabbit, 1:1000), GAPDH (Santa Cruz, V‐18, sc‐20357, goat, 1:2000), MYH11 (Sigma, M7786, mouse, 1:2000), MYLK (abcam, ab76092, rabbit, 1:2000), TGFB1I1 (Hic5, BD Transduction Laboratories, 61164, mouse, 1:3000), CNN1 (abcam, ab46794, rabbit, 1:5000), TAGLN (abcam, ab10135, goat, 1:3000), VCL (Sigma, V4505, mouse,1:5000). All HRP‐conjugated secondary antibodies were purchased from Abcam (Goat anti‐mouse IgG, Cat. No. ab205719; Goat anti‐rabbit IgG, Cat. No. ab205718; Donkey anti‐goat IgG, Cat. No. ab205723). Western blot images were acquired by ImageQuant LAS 8000 Imaging Station (GE).
9
Protein Expression Analysis in Lung Tissues
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Protein was isolated from the right lower lobe of lung tissues using a RIPA buffer (Beyotime, China) obeying the protocol of the manufacturer. Protein was then separated on 10% SDS-PAGE, transferred onto PVDF members (Millipore, USA), blocked with 5% nonfat milk, and probed with primary antibodies, such as PLCε-1 (ab109501, Abcam, USA), TLR4 (#14358, Cell Signaling Technology, USA), RelA (p65, #3039, Cell Signaling Technology, USA), caspase 3 (#9662, Cell Signaling Technology, USA), Bcl-2 (ab194583, Abcam, USA), Bax (ab32503, Abcam, USA), MLCK (ab76092, Abcam, USA), VE-cadherin (ab231227, Abcam, USA), occludin (ab216327, Abcam, USA), ZO-1 (ab191143, Abcam, USA), and GAPDH (ab8245, Abcam, USA) at 4°C overnight, respectively. Subsequently, the members were incubated with the secondary antibody goat anti-rabbit IgG HRP (ab6721, Abcam, USA) at room temperature for 1 h. Finally, the bands were visualized using an enhanced chemiluminescent agent (Bio-Rad, USA). The levels of protein were normalized using GAPDH.
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