Cells were lysed in cold RIPA buffer supplemented with protease inhibitors. Equal amounts of protein were resolved by 4–12% Bis-Tris/PAGE, transferred onto PVDF membranes, and blocked in buffer [5% (wt/vol) milk, TBST (TBS, pH 7.4, 0.05% Tween-20), 1 hr, room temperature]. Primary antibodies were diluted in blocking buffer, as follows: FUT8 (1:1,000; Proteintech, 66118), L1CAM, also referred as L1CAM Ab1 (1:5,000; Abcam, ab20148), L1CAM Ab2 (1:2000; Abcam, ab208155), L1CAM Ab3 (1:2000, Abcam, ab24345), GAPDH (1:5,000; Abcam, ab8245); NRP2 (1:5,000; Santa Cruz, sc13117), LAMB1 (1:1000; Abcam, ab44941), Integrin beta 1 (1:2000; Abcam, ab52971), ADAM10 (1:1000; Abcam, ab1997), α-Tubulin (1:5000; Sigma, T9026). Secondary antibodies were α-mouse or α-rabbit-HRP (1:5,000; Bio-Rad). Blots were developed by using Clarity™ Western ECL Blotting Substrate (BioRad) and imaged in LICOR Odyssey Fc imaging system.
Ab52971
Manufactured by Abcam
Sourced in United States, China, United Kingdom
Ab52971 is a laboratory reagent for research purposes. It is a recombinant protein that can be used in various experimental applications. No further details about its intended use or application are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab150361, by Abcam (4 mentions)
Ab52894, by Abcam (2 mentions)
Ab6124, by Abcam (2 mentions)
Ab23894, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab11575, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
α tubulin, by Merck Group (1 mentions)
Ab208155, by Abcam (1 mentions)
Ab24345, by Abcam (1 mentions)
Ab8245, by Santa Cruz Biotechnology (1 mentions)
Ab44941, by Abcam (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Solarbio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Culture media, by Thermo Fisher Scientific (1 mentions)
Aiib2, by Developmental Studies Hybridoma Bank (1 mentions)
Ab1416, by Abcam (1 mentions)
24 protocols using ab52971
1
Western Blot Analysis of Protein Expression
2
Integrin and Rac1 Regulation in Cell-ECM Interactions
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Antibodies used include anti-β1 integrin (ab52971, Abcam), anti-Rac1 (ab33186, Abcam), anti-E-cad (ab1416, Abcam), and anti-MUC-1 (ab109185, Abcam). Other reagents included β1 integrin inhibitor AIIB2 (Developmental Studies Hybridoma Bank, United States), type I collagenase (Solarbio, China), collagen gel (Nitta Gelatin, Japan), culture media (Gibco), and Lipofectamine 2000 and TRIzol (Invitrogen). siRNAs were synthesized by GenePharma, China, and the sequences were as follows: siRNA-β1 integrin: forward, 5′-GUU UAA UGU CUG GUG CUU TT-3′; reverse, 5′-AAG CAC CAG ACA UUA AAC TT-3′; siRNA-Rac1: forward, 5′-UAA AGA CAC GAU CGA GAA AUU-3′; reverse, 5′-UUU CUC GAU CGU GUC UUU AUU-3′; and siRNA-ctrl: forward, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; reverse, 5′-ACGUGACACGUUCGGAGAATT-3′.
3
Immunohistochemical Analysis of PDX Tissues
4
Immunophenotyping of Stem Cells by Flow Cytometry
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Cells were harvested by trypsinisation, centrifuged at 150 x g and resuspended in phosphate buffered saline (PBS). Non-specific binding was prevented by a 10% normal goat serum (NGS, Sigma) and 0.1% sodium azide incubation on ice. Primary antibodies were added to 1x105 cells per condition added for 30 minutes at room temperature. The antibodies used were against CD29 (ab52971, Abcam), CD44 (ab6124, Abcam), CD45 (ab10559, Abcam), CD73 (AD2, Miltenyi), CD90 (ab23894, Abcam), CD105 (43A4E1, Miltenyi) or relevant isotype controls. The cells were washed and incubated with the secondary antibodies (Cy5 anti-mouse or anti-rabbit, Eurogentec/Abcam) for 30 minutes at room temperature. The cells were washed, resuspended in 500 μl PBS with 3% NGS and analysed on a Beckman Coulter Cyan ADP with 635nm excitation. Fluorescence compensation and post-acquisition gating was performed using Summit V4.3.
5
Western Blot Analysis of Cell Signaling Proteins
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Cells were harvested in cold PBS and lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris at pH 7.5, 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl, 10 mM EDTA) containing protein inhibitor cocktail (Roche, Basel, Switzerland). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with primary antibodies and HRP-conjugated secondary antibody. β-Actin was used as a loading control. The antibodies used in this study were as follows: human anti-SHCBP1 (1:400; SAB1307183, Sigma-Aldrich, USA), anti-MYC (1:5,000; ab32072, abcam, USA), anti-EGFR (1:1,000; ab52894, abcam, USA), anti-KLF4 (1:1,000; ab129473, abcam, USA), anti-CD44 (1:1,000; ab51037, abcam, USA), anti-VEGFA (1:1,000; ab46154, abcam, USA), anti-CDK4 (1:500; 11026-1-AP, Proteintech, USA), anti-CCND1 (1:500; sc-8396, Santa Cruz Biotechnology, USA), anti-ITGA6 (1:400; sc-19622, Santa Cruz Biotechnology, USA), anti-ITGB1 (1:1,000; ab52971, abcam, USA), anti-ITGB5 (1:1,000; ab15459, abcam, USA), anti-COL4A2 (1:2,000; 55131-1-AP, Proteintech, USA), anti-GAPDH (1:2,000; sc-32233, Santa Cruz Biotechnology, USA), and HRP-conjugated secondary antibody (1:5,000; G-21040, Thermo Scientific, USA). The signals were detected by the Li-Cor Odyssey system (Li-Cor Biosciences, Lincoln, NE, USA).
6
Characterization of TMEM2-integrin interactions
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Recombinant soluble TMEM2 ECD (TMEM2–ECD), tagged at the C terminus with a 6× His epitope, was produced in HEK293 cells, as described previously (25 (link)). TMEM2–ECD was bound to ProBond nickel chelating resin (approximately 5 μg protein/20 μl resin; Thermo Fisher) by incubation for 2 h at 4 °C, and the resin was washed with HBSS++. Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 °C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDS-PAGE and immunoblotting with rabbit polyclonal anti-integrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal anti-integrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated). Detection of immunoreactive bands was carried out as described as previously.
7
Integrin β1 Expression Analysis
8
Integrin Expression in VSMCs Treated with FC-EVs
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VSMCs were placed in 96‐well plates and grown to 80% confluence and then treated with FBS‐free medium containing 20 μg/mL of FC‐EVs for 0 minutes, 15 minutes, 30 minutes, 1 hour, or 2 hours. The cells were washed with PBS and fixed with methanol for 5 minutes and then washed 3 times with PBS and blocked with 2% bovine serum albumin for 2 hours at 37°C. The integrins on the cell surfaces were evaluated using primary monoclonal antibodies for integrin β1 (ab52971; Abcam) or integrin α5 (ab150361; Abcam), followed by binding of an HRP‐conjugated secondary antibody (1:2000). The bound antibodies were quantified using a TMB peroxidase EIA substrate kit (Thermo Scientific), and the intensity of the color developed at an optical density at 450 nm was evaluated. Five parallel wells were set up for each condition.
9
Fibroblast Mechanotransduction Analysis
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At any given time after loading cyclic stretch, cultures of fibroblasts were harvested and lysed with cell lysis buffer containing l mmol/L phenylmethyl sulfonylfluoride (PMSF). After being resolved by standard sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE), cell lysates were electroblotted onto a polyvinylidene fluoride (PVDF) (Millipore, USA) membrane. Then, the membrane was incubated with different primary antibodies including rabbit anti‐human α‐smooth muscle actin primary antibodies (# ab5694, Abcam, UK), rabbit anti‐human integrin β1 (# ab52971, Abcam, UK), rabbit anti‐human lamin A/C (# ab108595, Abcam, UK), and rabbit anti‐human Nesprin‐2 (# ab217057, Abcam, UK). Next, an anti‐rabbit horseradish peroxidase‐conjugated secondary antibody (# CW01035, CWbiotech, China) was added. Following the supplier's instructions, chemiluminescent detection of proteins was performed with enhanced chemiluminescence (ECL) detection kit (GE Healthcare Amersham, China). The relative intensities of immunoreactive protein bands were quantified by scanning densitometry using ImageJ program inspired by NIH Image at Bethesda, Maryland.
10
Protein Expression Analysis in CRSwNPs
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Proteins were extracted from CRSwNPs tissues or cells using radioimmunoprecipitation assay (Beyotime Institute of Biotechnology), and the concentrations were determined according to the standard protocols of BCA protein assay kit (Beyotime Institute of Biotechnology), respectively. The total protein (30 µg/well) in the supernatant was separated via SDS-PAGE on 10% gel, and then transferred to PVDF membranes. After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with rabbit anti-MMP-2 (1:1,000; no. ab92536; Abcam), rabbit anti-MMP-9 (1:1,000; no. ab76003; Abcam), rabbit anti-TIMP-1 (1:1,000; no. ab211926; Abcam), rabbit anti-integrin β1 (1:1,000; no. ab52971; Abcam), rabbit anti-α-tubulin (1:5,000; no. ab18207; Abcam), rabbit anti-acetyl-α-tubulin (1:1,000; no. ab179484; Abcam) and rabbit anti-β-actin (1:5,000; no. ab8227; Abcam) antibodies. After three washes with TBS with 0.1% Tween-20, the immunoblots were incubated for 1 h at room temperature with goat alkaline phosphatase-labeled anti-rabbit antibody (1:1,000; cat. no. 14708; Cell Signaling Technology, Inc.). The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology). The blots were semi-quantified using ImageJ software (version 1.47; National Institutes of Health).
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