The whole eyeballs were immersed in 4% paraformaldehyde for 24 h at room temperature. Subsequently, the eyeballs were treated with gradient alcohol and xylene and embedded in paraffin for sectioning at a thickness of 4 μm. Tissue sections stained with hematoxylin/eosin were used to observe the corneal structure. Immunohistochemical staining was performed according to our previously published protocol52 (link). Briefly, antigen retrieval was performed with sodium citrate repair solution by microwave heating, non-specific antigens were eliminated by 3% H2O2, and the sections were blocked with goat serum for 1 h and incubated with primary antibodies against IL-1β (1:100, ab254360, Abcam), TNF-α (1:1000, ab183218, Abcam), TRAF6 (1:300, ab33915, Abcam), IRAK1 (1:1000, ab238, Abcam), and COX2 (1:400, ab179800, Abcam) overnight at 4 °C. Next day, the sections were incubated with the Envision horseradish peroxidase system (Gene Tech, Shanghai, China) for 2 h at room temperature. The sections were finally incubated with 3,3′-diaminobenzidine (DAB) (Gene Tech, Shanghai, China) for 5 min. The staining was observed and captured with an Eclipse Ni microscope (Nikon, Tokyo, Japan).
Ab238
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab238 is a laboratory instrument designed for general-purpose use in scientific research. The core function of this product is to provide a platform for various experimental procedures and analyses. No further details or interpretations are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab33915, by Abcam (4 mentions)
Ab16502, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab218130, by Abcam (3 mentions)
Ab86299, by Abcam (3 mentions)
Ab219413, by Abcam (2 mentions)
Ab76302, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab109526, by Abcam (2 mentions)
Ab32518, by Abcam (2 mentions)
Ab32536, by Abcam (2 mentions)
Eclipse ni microscope, by Nikon (1 mentions)
3 3 diaminobenzidine (dab), by Gene Tech (1 mentions)
Ab254360, by Abcam (1 mentions)
Ab183218, by Abcam (1 mentions)
Ab179800, by Abcam (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence ecl detection system, by Thermo Fisher Scientific (1 mentions)
Sc 365290, by Santa Cruz Biotechnology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
13 protocols using ab238
1
Immunohistochemical Analysis of Corneal Structure
2
Protein Expression Analysis in hRMECs
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Total proteins from hRMECs were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) containing protease inhibitors. Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime, Shanghai, China) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After sealed with 5% nonfat milk for 2 h, PVDF membranes were incubated with specific primary antibodies at 4°C overnight. Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20,000 dilutions; ab205718; Abcam, Cambridge, UK) were subsequently applied. Enhanced chemiluminescence (ECL) detection system (Thermo Scientific) was used for detection of immunoblots. ImageJ software (NIH, Bethesda, MD, USA) was used to analyze protein band grayscale normalized to β-actin. The primary antibodies are listed below: p-IκBα (1:1,000; #9246; Cell Signaling Technology, Danvers, MA, USA), IκBα (1:1,000; #4812; Cell Signaling Technology), p65 (0.5 μg/mL; ab16502; Abcam), PI3K (1:400; sc-365290; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-AKT (1:2,000; #4060; Cell Signaling Technology), AKT (1:1,000; #9272; Cell Signaling Technology), IRAK1 (1:1,000; ab238; Abcam), IRS1 (1:400; sc-8038; Santa Cruz Biotechnology), and β-actin (1:1,000; ab69512; Abcam).
3
Intestinal Tight Junction Protein Analysis
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The total proteins of intestinal tissues were extracted by RIPA protein lysis buffer (Invitrogen; USA), and the concentration and purity of proteins were measured by a BCA protein kit (Pierce; USA). Then, 40 μg of protein from each sample was separated by SDS–PAGE, transferred to PVDF membranes by the electric transfer method and blocked with skim milk at room temperature for 1 h. After incubation with primary and secondary antibodies, the protein bands were visualized by enhanced chemiluminescence (Pierce; USA). The primary antibodies were diluted at 1:1000 as follows: anti-ZO-1 (ab96587; Abcam; UK), anti-Occludin (ab216327; Abcam; UK), anti-TLR2 (ab213676; Abcam; UK), anti-Tollip (ab187198; Abcam; UK), anti-MyD88 (ab219413; Abcam; UK), anti-IRAK1 (ab238; Abcam; UK), anti-p-IRAK1 (ab218130; Abcam; UK), anti-p65 (ab16502; Abcam; UK), anti-p-p65 (ab76302; Abcam; UK), anti-MLCK (ab232949; Abcam; UK), and anti-GAPDH (ab8245; Abcam; UK). The grayscale value analysis was performed by using ImageJ software (version 1.52a; National Institutes of Health), and GAPDH was used as the loading control.
4
Western Blotting and Immunoprecipitation Assay
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For western blotting, total protein was extracted from the cultured cells using RIPA lysis and extraction buffer (Thermo Fisher Scientific) and separated on a 12% SDS–PAGE polyacrylamide gel.
The primary antibodies used in this study were IFITM3 (1:1,000 dilution, ab109429, Abcam), TRAF6 (1:100, sc-8409, Santa Cruz Biotechnology), IRAK1 (1:200, ab238, Abcam), p-ERK (1:800, #4376, Cell Signaling Technology), c-Fos (1:20,#4384, Cell Signaling Technology), c-Myc (1:100, 395R16, Cell Marque), and survivin (1:50, ab76424, Abcam). β-actin served as the internal control.
For the immunoprecipitation (IP) assay, the protein extracts from cultured cells were prepared using IP lysis buffer (50 mM Tris–Cl [pH 7.4], 0.5% NP-40, 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, 2 mM EGTA) supplemented with protease/phosphatase inhibitor mixtures (Roche), then centrifuged (12,000 g for 10 minutes at 4℃). The IP assay was performed as previously described [18 (link)].
The primary antibodies used in this study were IFITM3 (1:1,000 dilution, ab109429, Abcam), TRAF6 (1:100, sc-8409, Santa Cruz Biotechnology), IRAK1 (1:200, ab238, Abcam), p-ERK (1:800, #4376, Cell Signaling Technology), c-Fos (1:20,#4384, Cell Signaling Technology), c-Myc (1:100, 395R16, Cell Marque), and survivin (1:50, ab76424, Abcam). β-actin served as the internal control.
For the immunoprecipitation (IP) assay, the protein extracts from cultured cells were prepared using IP lysis buffer (50 mM Tris–Cl [pH 7.4], 0.5% NP-40, 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, 2 mM EGTA) supplemented with protease/phosphatase inhibitor mixtures (Roche), then centrifuged (12,000 g for 10 minutes at 4℃). The IP assay was performed as previously described [18 (link)].
5
Western Blot Analysis of Inflammatory Signaling
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Cells were rinsed in PBS and scraped in 400 ml of cell lysis buffer containing protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). Cell lysates were then centrifuged at 12,000 rpm for 15 min at 4°C. The protein concentration in each lysate was then determined using the BCA assay. SDS-PAGE gels were loaded with the same amount of protein, and the NC membrane was blocked for 30 min with skimmed milk. Several primary antibodies were used for western blotting (as 1:1000 dilutions): NF-κB p65, ab16502; IkBα, ab76429; TLR2, ab108998; MyD88, ab219413; IRAK1, ab238; TRAF6, ab33915; TIRAP, ab17218; TAK1, ab109526; p-NF-κB p65, ab76302; IRAK4, 4363; p-PI3K, ab182651 (Abcam, Cambridge, MA, United States). We also used β-actin, 4970, GAPDH, 5174; AKT, 9272; and p-AKT, 4060S (Cell Signaling Technology, United States). HRP-labeled goat anti-rabbit secondary antibodies were used at a dilution of 1:2000 (A0208; Beyotime Biotechnology, Shanghai, China) and immunoreactive bands were detected using an Amersham Imager 600 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
6
Western Blot Analysis of Cellular Proteins
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RIPA lysis buffer (Millipore, United States) was utilized for extracting the total protein from tissue specimens. By using the BCA protein quantification kit, the protein concentration was determined. After protein denaturation treatment, SDS–PAGE gel electrophoresis was used to separate cellular proteins. The electrophoresis was stopped according to the prestained marker band. The sample was then transferred to the PVDF membrane. Then, the membrane was blocked by 3% BSA prepared with TBS-T overnight at 4°C. After blocking, the membrane was incubated with diluted primary antibodies against MAP2K2 (1/1,000; ab265586; Abcam, United States), IRAK1 (1/1,000; ab238; Abcam, United States), RAC1 (1/1,000; ab97732; Abcam, United States), TRAF3 (1/1,000; ab23935; Abcam, United States), MAP3K7 (1/1,000; ab25879; Abcam, United States), SPP1 (1/1,000; ab166709; Abcam, United States), and GAPDH (1/1,000; ab8245; Abcam, United States) overnight at 4°C, followed by being incubated with goat anti-rabbit IgG–HRP antibody (Santa, United States) or goat anti-mouse IgG-HRP antibody (Santa, United States) for 2 h at room temperature. The protein was developed by ECL luminescent agent. ImageJ software was utilized for detecting the gray value.
7
Exploring Apoptotic Signaling Pathways in HK2 Cells
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HK2 cells were lysed in RIPA buffer (Beyotime), and the cell lysates were centrifugated at 14,000 g for 1 min. The supernatants were obtained, and protein concentration was detected with a BCA kit (Amyjet, Wuhan, China). A 20 µg protein sample was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane (Solarbio). The membrane was blocked in 5% bovine serum albumin (Solarbio). Subsequently, the membrane was incubated with antibody against B-cell lymphoma-2 (Bcl-2) (ab59348, 1 : 500 dilution, Abcam), Bcl-2-associated X (Bax) (ab263897, 1 : 2000 dilution, Abcam), cleaved caspase 3 (C-caspase 3) (ab2302, 1 : 1000 dilution, Abcam), IRAK1 (ab238, 1 : 2000 dilution, Abcam), or GAPDH (ab245355, 1 : 5000 dilution, Abcam) overnight and IgG labeled by horseradish peroxidase (HRP) (ab6721, 1 : 8000 dilution, Abcam) for 2 h. Next, the blots were interacted with ECL reagent (Yeasen), and then examined by Image J v1.8 software (NIH, Bethesda, MD, USA). GAPDH functioned as a loading reference, and relative protein expression was normalized to the control group.
8
IRAK1 Signaling Pathway Exploration
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LPS was purchased from Sigma-Aldrich (St. Louis, MO). PF was purchased from Yuanye (S31585, HPLC ≥98%, Shanghai, China). Antibodies against IRAK1 (ab238, polyclonal antibody), p-IRAK1 (ab218130, polyclonal antibody), IKKα/β (ab178870, monoclonal antibody), IκBα (ab32518, monoclonal antibody), p-IκBα (ab133462, monoclonal antibody), NF-κB (ab32536, monoclonal antibody), and p-NF-κB (ab86299, polyclonal antibody) were purchased from Abcam (Cambridge, UK). Antibodies against p-IKKα/β (2697, monoclonal antibody), α-tubulin (2144, polyclonal antibody), and anti-mouse/rabbit IgG (7076/7074, HRP-linked Antibody) were purchased from Cell Signaling Technology (Beverly, MA, USA). A chemiluminescent horseradish peroxidase (HRP) substrate was purchased from Millipore (Burlington, MA, USA).
9
Western Blot Analysis of Immune Signaling
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Total protein was obtained using RIPA lysis buffer, and approximately 30 µg of total protein in each sample were separated on SDS/PAGE gels in an electrophoresis system (Bio-Rad, Mini-Protein Tetra System) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with a TBST (TBS/0.15% Tween 20) solution containing 5% skim milk powder at room temperature for 1 h, the solution was discarded, and the PVDF membrane was washed three times with the TBST solution, followed by overnight probing at 4 °C with anti-Foxp3 (1:1000; Catalog No.: ab215206; Abcam, UK), anti-TRAF6 (1:5000; Catalog No.: ab33915; Abcam, UK), anti-IRAK1 (1:1000; Catalog No.: ab238; Abcam, UK), anti-p-IκB (1:1000; Catalog No.: 2859T; CST, USA), anti-p-p65 (1:1000; Catalog No.: 3033T; CST, USA), anti-STAT1 (1:1000; Catalog No.: 14994S; CST, USA), anti-p-STAT1 (1:1000; Catalog No.: 7649S; CST, USA), and anti-β-actin (1:5000; Catalog No.: 4970T; CST, USA) antibodies. The membrane was rinsed, incubated with diluted horseradish peroxidase (HRP)-labeled secondary antibody (1:50000; Catalog No.: 7071T; CST, USA) for 45 min at room temperature, and immunoreactive protein bands were detected using an enhanced chemiluminescence assay kit (Millipore). Images were acquired with a G-Box system (Syngene, Frederick, MD, USA) and analyzed with ImageJ software.
10
Protein Expression Analysis Protocol
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RIPA buffer containing 1% protease inhibitor was added to cells or tissues, which were subsequently lysed on ice for 30 mins. The lysed cells or tissues were then centrifuged for 20 mins, and the supernatants were collected. The total protein concentration in each supernatant was determined by the BCA method. Next, a 30 μg aliquot of protein from each sample was separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Millipore, Burlington, MA, USA), which were subsequently blocked with 5% skim milk for 1.5 hrs. Next, the membranes were washed and then incubated overnight at 4°C with the following primary antibodies (1∶1000, Abcam, Cambridge, UK): IRAK1 (Abcam, ab238), E-cadherin (Abcam, ab15148), N-cadherin (Abcam, ab18203), vimentin (Abcam, ab137321), IL-1β (Abcam, ab9722), NLRP3 (Abcam, ab214185), ASC (Abcam, ab227502), ERK1/2 (Abcam, ab17942), JNK1/2 (Abcam, ab112501), p-ERK1/2 (Abcam, ab223500), p-JNK1/2 (Abcam, ab4821), Caspase 3 (Abcam, ab32042), and GAPDH (Abcam, ab9485). The next day, the membranes were washed and then incubated with a 1∶5000 diluted secondary antibody (Abcam) for 2 hrs. The immunostained proteins were detected using an ECL substrate kit (Thermo Scientific, Waltham MA, USA), and results were displayed on a Bio-Rad Gel Doc/Chemi Doc Imaging System.
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