Radioimmunoprecipitation assay lysis buffer (Millipore, Billerica, MA, USA) was used to extract the total protein, as per the user's manual book. The operating procedures were in accordance with the report of Lai et al.20 The primary antibodies from Abcam (Cambridge, MA, USA) were exhibited as follows: anti‐B‐cell lymphoma‐2 (anti‐Bcl‐2; ab32124, 1:1000), anti‐Bcl‐2 associated X (anti‐Bax; ab32503, 1:1000), anti‐cleaved‐caspase‐3 (ab2302, 1:1000), anti‐CDK16 (ab15467, 1:1000), anti‐JAK2 (ab108596, 1:1000), anti‐phosphorylated‐JAK2 (anti‐p‐JAK2; ab32101, 1:1000), anti‐STAT3 (ab32500, 1:100), anti‐phosphorylated‐STAT3 (anti‐p‐STAT3; ab76315, 1:1000), and anti‐GAPDH (ab181602, 1:3000). After the incubation with anti‐rabbit IgG, HRP‐linked secondary antibody (ab205718, 1:5000), the protein blots were visualized on the membranes using ECL Ultra Western HRP Substrate (Millipore). Data analysis was completed by ImageLab software version 4.1 (Bio‐Rad, Hercules, CA, USA).
Ab205718
Manufactured by Merck Group
Sourced in Germany, Ireland, United Kingdom
The Ab205718 is a laboratory equipment product manufactured by Merck Group. It is designed for specific scientific applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information from the manufacturer would be required to present the core function of this product accurately without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Imagelab software version 4, by Bio-Rad (2 mentions)
Ab32500, by Abcam (1 mentions)
Ab108596, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Merck Group (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ecl ultra western hrp substrate, by Merck Group (1 mentions)
Ab2302, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Ab76315, by Abcam (1 mentions)
Ab32101, by Abcam (1 mentions)
Ab260043, by Abcam (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab7291, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Ab2302, by Merck Group (1 mentions)
4 protocols using ab205718
1
Protein Extraction and Western Blot
2
Protein Expression Analysis in LF Tissue
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A mixture of M-PER Tissue Protein Extraction Reagent (Pierce, IL, USA), EDTA, and protease inhibitors (Beyotime, Shanghai, China) was used to extract total protein from LF tissue samples, whereas LF cellular protein was isolated using RIPA lysis buffer containing protease inhibitors (Beyotime). A BCA kit (Beyotime) was then used to quantify protein levels in individual samples. Equal amounts of total protein from each sample (40 μg) were then separated via 10% SDS-PAGE and transferred onto PVDF membranes. Blots were then blocked with 5% SA, followed by overnight incubation with primary antibodies at 4° C. Antibodies were from Abcam (United Kingdom), were prepared in 5% BSA, and were as follows: anti-SIRT6 (#ab62739, 1:2000); anti-TGF-β1(#ab215715, 1:1000); anti-α-SMA (#ab7817, 1:3000); anti-Collagen I (#ab260043, 1:5000); anti-α-tubulin (#ab7291, 1:1000); and anti-β-actin (#ab8226, 1:5000). Following an additional 1 h incubation with HRP-conjugated secondary antibodies (#ab6789, 1:2000; #ab205718, 1:50000), protein bands were detected with an enhanced chemiluminescence kit (Millipore, Germany). The ImageJ software was then used for densitometric quantification, with α-tubulin or β-actin being used for normalization purposes.
3
Extracellular Vesicle Protein Profiling
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Total protein was extracted from small, medium and lEVs derived from 20 × 106 BMMCs (unstimulated, 100 ng/ml LPS and 0.5 μM A23187 stimulated), by sonication. Protease and phosphatase inhibitor cocktails were added (Sigma‐Aldrich). Protein samples (10–40 μg) and pre‐stained protein markers (26634, Spectra Multicolor Broad Range Protein Ladder) were separated by SDS‐PAGE and blotted onto 0.2 μm Immun‐Blot PVDF membranes (Bio‐Rad). Membranes were stained according to standard Western blot protocol using anti‐TSG101 (T5701, Sigma‐Aldrich), anti‐Alix (ab186429, Sigma‐Aldrich, Dublin, Ireland) and anti‐rabbit Abs (ab205718, Sigma‐Aldrich). Proteins were visualized with a chemiluminescent HRP substrate (32106, Thermo Scientific) and recorded by Gel documentation system (CHEMI Premium, VWR).
4
Protein Extraction and Western Blot Protocol
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Fresh protein lysis solution was produced by mixing RIPA Buffer (Sigma-Aldrich), protease inhibitor PMSF (Sangon), and phosphatase inhibitor Sodium orthovanadate (V) dodecahydrate (Sangon) with the ratio of 98:1:1 (v/v/v). Total proteins were extracted by adding the protein lysis solution for incubation on ice for 30 min and collecting the supernatant through centrifugation at 7000 rpm/min for 15 min. The operating steps for Western blot followed the detailed descriptions published previously.21 (link) The antibodies were all bought from Abcam (Cambridge, UK): anti-proliferating cell nuclear antigen (anti-PCNA; ab18197, 1:1000), anti-Cyclin D1 (ab226977, 1:1000), anti-total-caspse3 (anti-t-caspase 3; ab4051, 1:500), anti-cleaved caspase 3 (anti-C-caspase 3; ab2302, 1:1000), anti-matrix metalloproteinase 9 (anti-MMP9, ab38898, 1:1000), anti-NOVA2 (Sigma-Aldrich, AV40399, 1:1000), anti-GAPDH (ab9485, 1:1000) and Goat Anti-Rabbit IgG H&L (HRP) second antibody (ab205718, 1:3000). GAPDH acted as the internal control in this assay. ImageLab software version 4.1 (Bio-Rad, Hercules, CA, USA) was exploited to perform the grey level analysis.
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