The cells were lysed for 20 min in 180 μL of lysis buffer (Beyotime, Shanghai, China) on ice before the centrifugation at 4 °C and 14,000 rpm for 10 min. The total protein concentration was estimated using a bicinchoninic acid (BCA) protein detection kit (CWBIO, Shanghai, China) with bovine serum albumin (BSA) as the standard. The proteins were separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After being blocked with 5% (w/v) skimmed milk for 2 h, the membranes were probed with corresponding primary antibodies overnight at 4 °C and then incubated with secondary antibodies conjugated with horseradish peroxidase for 2 h. The primary antibodies used were: TLR4 (1:1000, Abcam, ab13556), myeloid differentiation factor 88 (MyD88, 1:1000, Abcam, ab133739), nuclear factor kappa-B (NF-κB) p65 (1:1000, Abcam, ab32536), NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), claudin-1 (1:2000, Abcam, ab211737), occludin (1:1000, Abcam, ab216327), ZO-1 (1:1000, Abcam, ab276131), myosin light-chain kinase (MLCK, 1:1000, ABclonal, A3835), and β-actin (1:5000, Abcam, ab179467). The protein intensity was quantified using the Image J software (version 1.8.0, NIH, Bethesda, MD, USA).
Ab133739
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab133739 is a laboratory reagent produced by Abcam for research use. It is a polyclonal antibody raised in rabbit against a synthetic peptide derived from the C-terminus of the human alpha-synuclein protein. This antibody is designed to detect and identify alpha-synuclein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab13556, by Abcam (6 mentions)
Ab32536, by Abcam (5 mentions)
Ab8226, by Abcam (4 mentions)
Ab22048, by Abcam (4 mentions)
Ab32503, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Lysis buffer, by Beyotime (2 mentions)
Ab211737, by Abcam (2 mentions)
Ab76302, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab86299, by Abcam (2 mentions)
Ab208113, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab32042, by Abcam (2 mentions)
Ab207297, by Abcam (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ab233706, by Abcam (2 mentions)
Ab178945, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
β actin, by Merck Group (2 mentions)
21 protocols using ab133739
1
Western Blot Analysis of Tight Junction Proteins
2
Western Blot Analysis of Protein Expression
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Total proteins were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the concentration of the protein samples was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Proteins were subjected to SDS-PAGE and then electrophoretically transferred onto PVDF membranes. Membranes were incubated with primary antibodies overnight at 4°C after blocking in 5% nonfat milk in TBST buffer, which was then coincubated with horseradish peroxidase (HRP)-conjugated anti-mouse/anti-rabbit IgG (Thermo Fisher Scientific). Proteins were visualized using a detection system of enhanced chemiluminescence (ECL), and the bands were analyzed using BandScan ImageJ software. The primary antibodies used in this study were as follows: anti-CHOP (diluted 1 : 1000, ab11419, Abcam), anti-caspase 12 (diluted 1 : 1,000, ab62484, Abcam), anti-GRP78 (diluted 1 : 100, ab21685, Abcam), anti-NLRP3 (diluted 1 : 500, ab214185, Abcam), anti-ASC (diluted 1 : 1000, ab155970, Abcam), anti-pro-IL-1β (diluted 1 : 500, ab2105, Abcam), anti-IL-1β (diluted 1 : 1500, ab9722, Abcam), anti-p-NF-κB (p-p65) (diluted 1 : 2000, ab86299, Abcam), anti-NF-kB p65 (diluted 1 : 300, ab19870, Abcam), anti-TLR4 (diluted 1 : 500, ab13556, Abcam), anti-MyD88 (diluted 1 : 1000, ab133739, Abcam), and anti-β-actin (diluted 1 : 1000, ab8226, Abcam). β-Actin was used as an internal control.
3
Protein Expression Analysis of Inflammatory Mediators
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The total protein was extracted utilizing RIPA buffer (Solarbio), separated via SDS-PAGE gel (10%) and transferred to PVDF membrane (Beyotime). Subsequently, 5% slim milk was used to block membranes under indoor environment for 1 h, followed by reaction with primary antibodies against B-cell lymphoma-2 (Bcl2, 26 kDa, ab182858, 1:1000, Abcam), Bcl-2-associated X protein (Bax, 21 kDa, ab32503, 1:1000, Abcam), IL-6 (23 kDa, ab208113, 1:1000, Abcam), IL-1β (31 kDa, ab283818, 1:1000, Abcam), TNF-α (26 kDa, ab6671, 1:1000, Abcam), IL-10 (20 kDa, ab33471, 1:1000, Abcam), medullary differentiation protein 88 (Myd88, 33 kDa, ab133739, 1:5000, Abcam), Phospho-nuclear factor kappaB (NF-kB) p65 (p-p65, 65 kDa, 3033T, 1:1000, Cell Signaling Technology, Danvers, Massachusetts, USA), NF-kB inhibitor alpha (IκB-α, 36 kDa, AI096, 1:1000, Beyotime), Cleaved-caspase9 (35 kDa, 9505T, 1:1000, Cell Signaling Technology), and internal reference GAPDH (36 kDa, ab181602, 1:10000, Abcam) overnight at 4℃. Subsequently, Rabbit IgG H&L (HRP) secondary antibody (ab205718, 1:10000, Abcam) or Rat IgG(H + L) (HRP) secondary antibody (SA00001-15, 1:2000, proteintech, Wuhan, China) was applied to interact with the membrane for 2 h in indoor environment. Thereafter, BeyoECL Star Kit (Beyotime) was used to visualize the immunoblots.
4
Analyzing Myd88 Protein Expression
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The total protein was extracted with the Radio Immunoprecipitation Assay kit (R0010, Beijing Solabio Life Sciences Co., Ltd., Beijing, China), and the protein concentration was determined using the bicinchoninic acid protein assay kit (GBCBIO Technologies Co., Ltd., Guangzhou, China). A total of 40 µg of each sample was loaded, electrophoresed with 6%, 10%, and 15% sodium dodecyl sulphate-Xpolyacrylamide gel electrophoresis, and transferred to the polyvinylidene fluoride membrane (Millipore, MA). Following blockage using Tris-buffered saline Tween solution with 5% bovine serum albumin at ambient temperature, the membrane was reacted with diluted primary antibody to Myd88 (ab133739, 1:2000, Abcam Inc., Cambridge, UK) overnight at 4 ºC as well as with secondary antibody of goat anti-rabbit immunoglobulin G (IgG, ab97051, 1 : 2000, Abcam) at room temperature for 2 hours. Then, the Western blots were developed by electrochemiluminescence and imaged on Image Quant LAS 4000C (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
5
Western Blot Analysis of Apoptosis Markers
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The cell or tissue sample was lysed with an RIPA lysis buffer containing 1 mmol/L PMSF. Then, the homogenates were prepared and centrifuged at 12,000 rpm for 10 min at 4°C. The protein concentration was determined by the BCA method. Non-specific binding was blocked with 5% skimmed milk for 1.5 h, and the membranes were incubated with diluted primary antibodies against Caspase-3 (ab32042, 1:800; Abcam, Cambridge, MA, United States), TLR4 (ab13556, 1:800; Abcam), MyD88 (ab133739,1:800; Abcam), NF-κB p65 (ab207297, 1:800; Abcam), and β-actin (ab8226, 1:1,000; Abcam) overnight at 4°C and in the secondary antibody for 1 h at room temperature. The signals were determined by the Amersham prime ECL Plus detection system (Pittsburgh, PA).
6
Protein Extraction and Western Blot
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The cells were lysed in equal volumes of ice-cold lysis buffer containing a protease inhibitor cocktail (Pierce Chemical Co.). Cell homogenates were boiled for 5 min in 5× Laemmli sample buffer. Total proteins were extracted with a Whole Protein Extraction kit (KGP250, KeyGEN BioTECH, China). Polyvinylidene difluoride membranes were then blocked in 5% fat-free milk or 5% BSA in TBS containing 0.05% Tween 20. Following overnight incubation at 4 °C with antibodies targeting TLR5 (ab13876, Abcam, UK), MyD88 (ab133739, Abcam, UK), p65 (ab32536, Abcam, UK), phosphorylated p65 (p-p65, ab86299, Abcam, UK) and GAPDH (ab181602, Abcam, UK), the membranes were incubated with HRP-conjugated secondary antibodies (KGAA35, KGAA37, KeyGEN BioTECH, China) at a 1:150,000 dilution for 1 h at room temperature and developed with a SuperSignal chemiluminescent detection system.
7
Western Blot Analysis of TLR4 and MyD88
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For total protein extraction, RIPA lysis buffer (KGP2100, Keygen Biotech, Shanghai, China) with a protease inhibitor cocktail (KGP2100, Keygen Biotech, Nanjing, China) and a phosphatase inhibitor mixture (KGP2100, Keygen Biotech, Nanjing, China) were utilized to lyse the tissues. The protein concentrations were detected using the BCA Protein Assay Kit (KGP902, Keygen Biotech, Nanjing, China), and Western blotting was performed using sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). The transferred membranes were incubated at 4 °C overnight with the following primary antibodies: mouse anti-TLR4 antibody (1:1000, 16c5074; Abcam, Cambridge, UK), rabbit anti-MyD88 (1:1000, ab133739; Abcam, China) and rabbit anti-β actin (1:1000, ab8226; Abcam, Cambridge, UK). The membranes were then incubated with the corresponding secondary antibodies, including HRP antirabbit antibody (1:10,000, SA00001-1/SA00001-2, Proteintech, Chicago, IL, USA), for 1 h at room temperature. The blots were visualized using ECL chemiluminescent liquid (KGP1127, Abcam, Cambridge, UK) and an ultrasensitive chemiluminescence instrument (Bio-Rad, Hercules, CA, USA), and the densities were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).
8
Immunofluorescence Staining of pAECs
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pAECs grown on collagen type I-coated coverslips in six-well plates were washed with PBS 1X before fixation with 4% paraformaldehyde (Electron Microscopy Sciences, 15710; can be stored at −80 °C). After permeabilization in PBS 1X/FBS 10%/Triton 0.1% for more than 90 min, the primary antibody against RAGE (1/1000, ab37647, Abcam, Paris, France), HMGB1 (1/400, ab79823, Abcam, Paris, France), Myd88 (1/250, ab133739, Abcam, Paris, France), TIRAP (1/100, ab17218, Abcam, Paris, France), Diaphanous-1 (1/400, ab11173, Abcam, Paris, France), and p65 NF-κB (1/400, 8242, Cell signaling, Saint-Cyr-L’Ecole, France) was applied overnight at 4 °C. After three washes in the permeabilization buffer, the secondary antibody, anti-rabbit Alexa Fluor® 488 (1/1000, A21206, Life Technologies, Villebon-Sur-Yvette, France), was incubated for 2 h at room temperature. Slides were washed three times in PBS 1X and incubated with Hoescht (15 min, dilution in PBS 1X 1/10,000; bisBenzimide H, 33258, Sigma-Aldrich, Saint-Quentin-Fallavier, France). Finally, slides were mounted with CitiFluor™ Tris-MWL 4-88 (Electron Microscopy Science, Hatfield, PA, USA) and examined under a Zeiss LSM800 Airyscan for cells. For the negative controls, incubation without the primary antibody was performed.
9
Western Blot Analysis of Synaptic and Inflammatory Proteins
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The tissues were homogenized in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) and supplemented with complete EDTA-free protease inhibitor cocktail and PhosSTOP Phosphatase Inhibitor. The protein samples were mixed with 5 × dual color protein loading buffer (Fudebio, Hangzhou, China) and boiled at 98 °C for 10 min. Equal amounts of protein (30 μg/lane) were loaded on a SDS-PAGE gel at a constant voltage and then transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). After being blocked and incubated with the primary and secondary antibodies, the bands were visualized using a chemiluminescence image analysis system (Tanon, Shanghai, China) with FDbioFemto ECL (Fudebio, Hangzhou, China). The band intensities were quantified using Gel-Pro Analyzer software (Media Cybernetics, Maryland, USA). The primary antibodies were included the following: synaptosomal associated proteins 25 (SNAP-25; Abcam, ab109105), postsynaptic density proteins 95 (PSD-95; CST, 3450), Ibα1 (Abcam, ab178846), TNF-α (Abcam, ab215188), IL-6 (Abcam, ab233706), NLRP3 (Abcam, ab263899), IL-18(Abcam, ab243091), IL-1β (Abcam, ab254360), caspase-1 (Abcam, ab207802), TLR4 (Abcam, ab22048), myeloid differentiation factor 88 (MyD88; Abcam, ab133739), NF-κB p65 (Abcam, ab32536), GAPDH (ABclonal, AC033), ZO-1 (ABclonal, A11417), occludin (ABclonal, A12621) and β-actin (ABclonal, AC026).
10
Hippocampal TLR4 Signaling Pathway
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Radioimmunoprecipitation buffer containing a protein phosphatase inhibitor (Sigma-Aldrich) was used to lyse the left hippocampal tissues of rats from each group (n = 5). After extracting the total proteins, they were transferred to a polyvinylidene fluoride membrane, blocked for 2 h, and incubated with primary antibodies (1:1,000; Abcam) against TLR4 (ab217274), MyD88 (ab133739), and TRAF6 (ab33915), followed by appropriate secondary antibodies. To visualize blots, an ultra-sensitive FujiFilm LAS 4000 chemiluminescent liquid-based imaging analyzer was used. The relative intensities of individual bands were analyzed using ImageJ.
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