Immunoblotting was carried out as described previously [11 (link)]. The following antibodies were purchased from Abcam: anti-GAPDH (1:2500, 37 kDa, AB9485, RRID:AB_307275), anti-IL-6 (1:2000, 17 and 50 kDa, ab9324, RRID:AB_307175), and anti-ICAM-1 (1:4000, 90 kDa, AB53013, RRID:AB_870702). Anti-ACE2 (1:1000, 125 kDa, MA532307, RRID:AB_2809589) and anti-HIF-1α (1:1000, 130 kDa, PA5-85,494, RRID:AB_2792634) antibodies were obtained from Thermo Scientific. The following antibodies were purchased from Cell Signaling Technology: anti-ERK1/2 (1:1000, 42, 44 kDa, #4695, RRID:AB_390779), anti-phospho-ERK1/2 (Thr202/Tyr204), (1:1000, 42, 44 kDa, # 9101, RRID:AB_331646), anti-MSK1 (1:1000, 95 kDa, #3489, RRID:AB_2285349), anti-phospho-MSK1 (Thr581) (1:1000, 95 kDa, #9595, RRID:AB_2181783), anti-p38 (1:1000, 43 kDa, #9212, RRID:AB_330713), anti-phospho-p38 (Thr180/Tyr182) (1:1000, 43 kDa, #4511, RRID:AB_2139682), anti-phospho-JNK (Thr183/Tyr185) (1:1000, 46, 54 kDa, #4668, RRID:AB_823588), anti-JNK (1:1000, 46, 64 kDa, #9252, RRID:AB_2250373), anti-phospho-NFκB p65 (Ser536) (1:1000, 70 kDa, #3033, RRID:AB_331284), and anti-NFκB p65. (1:1000, 70 kDa, #6956, RRID:AB_10828935). The following antibody were obtained from ABclonal (Woburn, MA): anti-phospho-NFkB p65 (S276) (1:1000, 70 kDa, AP0123, RRID:AB_2771505). An anti-phospho-MBP antibody (1:200, 18 kDa, 13–104) was purchased from Merck.
Anti icam 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-ICAM-1 is a laboratory reagent that detects the presence of the Intercellular Adhesion Molecule 1 (ICAM-1) protein. ICAM-1 is a cell surface glycoprotein involved in the adhesion of cells to other cells. This reagent can be used to identify and quantify ICAM-1 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti vcam 1, by Abcam (13 mentions)
Anti gapdh, by Abcam (5 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti bax, by Abcam (3 mentions)
Anti β actin, by Abcam (3 mentions)
Anti mmp9, by Abcam (3 mentions)
Anti vcam 1 antibody, by Abcam (2 mentions)
Vcam 1, by Abcam (2 mentions)
Anti cyclin b1, by Abcam (2 mentions)
Anti txnrd1, by Abcam (2 mentions)
Anti cleaved parp, by Abcam (2 mentions)
Anti cleaved caspase 3, by Abcam (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
Secondary detection antibody, by Abcam (2 mentions)
Anti e cadherin, by Abcam (2 mentions)
Anti vimentin, by Abcam (2 mentions)
Anti p21, by Abcam (2 mentions)
Anti p smad3, by Abcam (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Anti cyclin d1, by Abcam (2 mentions)
29 protocols using anti icam 1
1
Immunoblotting Antibody Validation Protocol
2
Western Blot Analysis of Lung Tissue Proteins
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Lung samples were homogenized in RIPA buffer (AR0101-100, Boster, Wuhan, China) with a protease inhibitor cocktail. The protein concentration was measured by the BCA method (Thermo Scientific, MA, USA). Samples containing 30 μg of denatured total protein were separated by 10% SDS-PAGE and then transferred to 0.45 μm PVDF membranes. The membranes were blocked with 5% nonfat dry milk for 1 hour and incubated overnight at 4°C with the following antibodies: anti-ICAM-1 (dilution, 1 : 1000, Abcam, Cambridge, Massachusetts, USA), anti-P65 (dilution, 1 : 1000, CST, USA), anti-IκBα (dilution, 1 : 2000, HuaBio, Hangzhou, China), anti-p-P65 (dilution, 1 : 1000, CST, USA), anti-COX-2 (dilution, 1 : 1000, HuaBio, Hangzhou, China), and anti-β-actin (dilution, 1 : 1000, Bioss, Beijing, China). HRP-conjugated secondary antibodies (dilution, 1 : 5000, BA1054, Boster, Wuhan, China) were incubated for 1 hour at room temperature. Then, proteins were detected by chemiluminescence reagent (AR1171, Boster, Wuhan, China). The gray value of the band was observed and analyzed using the ImageJ software.
3
Protein Expression Analysis of Cell Signaling Pathways
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After treatment, cells were washed 3× with ice-cold Hank's Balanced Salt Solution (HBSS) and lysed with Radioimmunoprecipitation Assay buffer (RIPA) buffer containing protease inhibitors (Boston Bioproducts BP-421) Son et al., 2013 (link). The protein content of each sample was determined by Pierce BCA protein assay. Aliquots of cell lysate were resolved on 10% to 12% sodium dodecyl sulfate–polyacrylamide gels and subsequently transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with the following primary antibodies: anti-KLK10 (BiossUSA bs-2531R, 1:1000), anti-GAPDH (Abcam ab23565, 1:2000), anti-β-actin (Sigma-Aldrich A5316, 1:2000), anti-VCAM1 (Abcam ab134047, 1:1000), anti-ICAM1 (Abcam ab53013, 1:1000), and anti-phospho-NFκB p65 S356 (Cell Signaling #3033, 1:1000) overnight at 4°C in 5% milk in TBST at the concentration recommended by the manufacturer, followed by secondary antibody addition for 1 hr at RT in 5% milk in TBST. Protein expression was detected by a chemiluminescence method (Son et al., 2013 (link)).
4
Evaluating Leukocyte Transmigration in DSS-Induced Colitis
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To test whether an increased hemovascular leukocyte transmigration, rather than a reduced lymphovascular leukocyte clearance, could be responsible for the aggravated inflammatory phenotype and amplified intestinal immune cell burden seen in DSS-treated FoxC2(+/−) mice, the colonic expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) was evaluated in DSS-treated WT and FoxC2(+/−) mice. Colon samples were prepared as described above, immunoblotted to either nitrocellulose (ICAM-1) or polyvinyl difluoride membranes (VCAM-1) and incubated overnight with anti-ICAM-1 (1 µg/mL, Abcam) or anti-VCAM-1 antibodies (1:2000, Abcam). Band intensity (densitometry) relative to actin was quantified using NIH Image-J analysis program (imagej.net ).
5
Endothelial Cell Surface Protein Expression
6
Western Blot Analysis of Cell Adhesion
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Western blot analysis was performed as described previously [32 (link)], with minor modifications. Briefly, aliquots of 50 μg of protein were subjected to 7.5% SDS-PAGE and transferred onto Hybond-P + polyvinylidene difluoride membranes (Amersham Biosciences UK Ltd). The membranes were incubated with primary antibodies; anti-ICAM-1, -VCAM-1, -P2Y2R (Santa Cruz) or -phosphor-VE-cadherin (Y658) (Abcam, Cambridge, UK). β-Actin (Sigma Aldrich, St Louis, MO, USA) was used as loading control for normalization to the protein expression.
7
Protein Expression Analysis in Esophageal Cancer Cells
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The total protein in transfected KYSE450 and KYSE510 cells was extracted and quantified with the RIPA buffer (Solarbio, China), which was supplemented with 1% proteinase inhibitor cocktail. The protein concentration was obtained with the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). Next, the quantified protein was loaded into 10% or 12% SDS-PAGE for electrophoresis separation. The separated protein bands were then electronically transferred to the membranes (Sigma-Aldrich, USA). Following that, the membranes were put under ice for 2 h with 5% BSA at room temperature. This step was followed by the incubation of the membranes overnight with primary antibodies at 4 °C and 1.5 h incubation with secondary detection antibody (Cat# ab205718, Abcam, USA) at room temperature. Finally, the protein immunoblots were visualized using the Enhanced Chemiluminescent (ECL) Reagent Kit (Thermo Fisher Scientific. USA). The density of the blots was obtained using ImageJ software. All the primary antibodies were purchased from Abcam (USA), including anti-CyclinB1 (Cat# ab32053), anti-ICAM1 (Cat# ab53013), anti-VCAM1 (Cat# ab134047), anti-Cleaved PARP (Cat# ab32561), anti-Bax (Cat# ab32503), anti-Cleaved Caspase-3 (Cat# ab2302), anti-TXNRD1 (Cat# ab124954) and anti-β-actin (Cat# ab8227).
8
Western Blot Analysis of EMT Markers
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HeLa cells were lysed using the Protein Extraction kit according to the manufacturer's protocols (Beyotime Biotechnology). Total proteins (30 μg) were separated by 10% SDS‐PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% non‐fat milk in Tris‐buffered saline containing Tween‐20 for ~2 hours at room temperature, prior to incubation with the primary antibodies. The membranes were probed with anti‐E‐cadherin (1:2000; Abcam), anti‐ICAM1 (1:2000; Abcam), anti‐Vimentin (1:2000; Abcam), anti‐TGFBR1 (1:1000; Abcam), anti‐p‐ERK (1:2000; Abcam), anti‐ERK (1:2000; Abcam), anti‐SMAD2 (1:3000; Abcam), anti‐p‐SMAD2 (1:2000; Abcam), anti‐SMAD2 (1:3000; Abcam), anti‐p‐SMAD3 (1:1000; Abcam), anti‐SMAD3 (1:2000; Abcam), anti‐p21 (1:3000; Abcam) and anti‐GAPDH (1:3000; Abcam) antibodies overnight at 4°C. Subsequently, membranes were incubated with HRP‐conjugated secondary antibody (1:3000; Cell Signaling Technology, Danvers, MA, USA) for 1 hour at 37°C. An ECL system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to detect the immunoreactive bands. Relative protein expression levels were normalized to that of GAPDH. Protein expression levels were measured using Image Pro Plus software v.6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
9
AEE Crystal Extraction and Assay
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Transparent AEE crystals with a purity of 99.5% according to RE-HPLC were prepared in the Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS. The H2O2 solution (cat number: 323381), dimethyl sulfoxide (DMSO) and Trypsin-EDTA were supplied by Sigma (St. Louis, MO, USA). Deionized water (18.25 MΩ) was prepared with a Direct-Q®3 system (Millipore, Bedford, MA, USA). The Annexin V/PE apoptosis detection kit was purchased from BD Biosciences (San Jose, CA, USA). Anti-ICAM-1, anti-VCAM-1, and anti-E-selectin antibodies were obtained from Abcam (Cambridge, MA, USA). The rat-soluble vascular cell adhesion molecule 1(sVCAM-1) ELISA kit, rat soluble intercellular adhesion molecule 1 (sICAM-1) ELISA kit, and rat soluble E-selectin (sE-selectin) ELISA kit were obtained from Elabscience (Wuhan, China); the advanced glycation end product (AGE) ELISA kit was purchased from Wuhan USCN Business Co., Ltd. (Wuhan, China). MS-grade acetonitrile was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Carboxymethylcellulose sodium (CMC-Na) was supplied by Tianjin Chemical Reagent Company (Tianjin, China)
10
Optimized Immunohistochemistry Staining Protocol
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Staining was performed using a Discovery Ultra Autostainer (Ventana). For each immunohistochemistry (IHC) Ab, positive control tissue was initially used for optimization and identification of staining morphology, typically with archival paraffin-embedded tonsil, thymus, bone marrow, or spleen. Optimal Ag retrieval, primary Ab dilution, and detection kits were then determined by staining 4-µm sections of intestine. All Ag retrieval was optimal using a Standard Cell Conditioning 1 buffer (Ventana) retrieval protocol for 64 min at 100°C. The following anti-human Abs were optimized: anti-pSMAD3 (phosphor-S423 and S425, rabbit monoclonal clone EP823Y, dilution 1:100; Abcam), anti–MAdCAM-1 (mouse monoclonal, clone 355G8; Thermo Fisher Scientific, dilution 1:100), anti–ICAM-1 (rabbit monoclonal, clone EPR4776, dilution 1:400; Abcam), and anti–VCAM-1 (rabbit monoclonal, clone EPR5047, dilution 1:250; Abcam). The Ultraview DAB Detection Kit (Ventana) was used for all Abs. Digital images were acquired using a Vectra 3 Slide Scanner (PerkinElmer) for cell adhesion molecules and an BX43 microscope (Olympus) for pSMAD3 IHC. Unless otherwise noted, representative images were selected from 10 samples stained in each group.
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