In order to prepare the protein sample, the frozen jejunum tissue was lysed using tissue protein lysis buffer, and the protein concentration was determined according to the instructions of the bicinchoninic acid (BCA) protein quantitative kit (Sun Bio, Beijing, China). Sodium dodecyl sulfate–polyacrylamide gels (10–12%) were configured according to the weight of detected proteins. Fifty micrograms of protein in each sample was extracted to perform electrophoresis, transferred to nitrocellulose filter membranes, then was incubated overnight at 4°C with primary antibodies against occludin (1:1,000, ab216327), ZO-1 (1:1,000, ab96587), Rho (1:1,000, ab40673), ROCK1 (1:2,000, ab45171), and β-actin (1:2,000) (all from Abcam, Cambridge, MA, USA). After being washed three times, the membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc. USA) for 2 h at 37°C. Then the membranes were reacted using enhanced chemiluminescence (ECL) solution (Millipore, Corporation, Billerica, MA, USA), and then were scanned (Konica Minolta Medical Imaging, Inc., Wayne, NJ, USA). The expression of protein was quantitatively analyzed using target protein/β-actin with Adobe Photoshop (Adobe, Mountain View, CA, USA) and Lab Works (UVP, Upland, CA, USA) software.
Ab45171
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab45171 is a primary antibody used for immunohistochemistry and western blotting applications. It targets a specific protein of interest. The product details and specifications are available on the Abcam website.
Automatically generated - may contain errors
Lab products found in correlation
Ab71598, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab6721, by Abcam (3 mentions)
Ab205718, by Abcam (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
G8795, by Merck Group (2 mentions)
Ab129002, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab125025, by Abcam (2 mentions)
Sc 179, by Santa Cruz Biotechnology (2 mentions)
Ab181602, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab187027, by Abcam (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Ab216327, by Abcam (1 mentions)
Ab96587, by Abcam (1 mentions)
Ab40673, by Abcam (1 mentions)
Bradford assay dye reagent, by Bio-Rad (1 mentions)
30 protocols using ab45171
1
Protein Expression Analysis in Jejunum Tissue
2
ROCK1/2 Expression in HUVECs under GHS Conditions
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The HUVECs on Flat, HP1, HP2, and HP3 GHS for 2 days were washed twice with PBS and disrupted with 1 × cell lysis buffer (9803, Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma). A quantitative analysis of the samples was performed using the Bradford assay dye reagent (500–0006, Bio-Rad). The sample protein (10 μg) was boiled in 1 × loading dye for 5 min and subjected to electrophoresis in a 10% polyacrylamide gel with sodium dodecyl sulfate. After transfer to a polyvinylidene fluoride membrane (ISEQ. 00010, Millipore), the membranes were blocked with 5% bovine serum albumin containing 1 × TBST (a mixture of Tris-buffered saline and Tween 20; WH400028806, 3 M) at RT for 1 h. The membranes were incubated with anti-ROCK1 (1:1000; ab45171, Abcam), anti ROCK2 (1:1000; ab71598, Abcam), and anti-GAPDH (1:2000; G8795, Sigma) antibodies at RT for 2 h. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (1:3000; Santa Cruz) in TBST at RT for 1 h. Chemiluminescence was visualized with the ECL Plus reagent (32132, Thermo Fisher Scientific) and recorded on X-ray film.
3
Picroside II Attenuates Oxidative Stress
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The following reagents were used in the study: picroside II (CAS No. 39012-20-9, C23H28O13, 512.48, Tianjin Kuiqing Med. Tech. Co. Ltd.); apocynin (CAS No. 498-02-2, C9H10O3, 166.17, Sigma Aldrich); trans-4-bromine cinnamon acid (TBCA, CAS No. 1200-07-3, C9H7BrO2, 227.05, Sigma Aldrich); 2,3,5-triphenyl tetrazolium chloride (TTC, Chinese Chemical Reagent Co., Ltd.); phenylmethylsulfonyl fluoride (PMSF, no. 329-98-6, Beijing Solarbio Tech. Co. Ltd.); an enhanced BCA protein assay kit (No. P0010, Beyotime Institute of Biotech., China); mouse anti-Rac1 monoclonal antibody (Abcam, ab33186), rabbit anti-Nox2/gp91phox monoclonal antibody (Abcam, ab129068), rabbit anti-ROCK1 monoclonal antibody (Abcam, ab45171), anti-MMP2 (Abcam, ab92536), anti-MLCK (Abcam, ab76092), anti-claudin-5 (Santa Cruz Biotech., Lot# L2013); goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (AB136817, Abcam, USA); rabbit anti-β-actin antibody (BA2305, Wuhan Boster Biological Co., Ltd.); rat NADPH oxidase ELISA kit (RG3022) and rat ROS ELISA kit (RG3054) form Trust Specialty Zeal; and Evans Blue (EB, CAS:314-13-16, Sigma Aldrich).
4
Immunoprecipitation of Ubiquitin in CaOV3 Cells
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Control, LINC00452- overexpressing and knockdown CaOV3 cells from 10-cm culture dishes were lysed with RIPA buffer, respectively. Human IgG (Bioss, #bs-0297P, 1:150) or ROCK1 antibody (Abcam, #ab45171, 1:50) were incubated separately with each cell lysate overnight at 4°C with gentle rotation. 20 μl Protein A/G agarose beads (Beyotime Biotechnology, #P1012) were added and incubated at 4°C with gentle rotation for 2 h. After the sequential wash with PBS and cell lysis buffer, the agarose beads were resuspended in 20 μl 1x SDS-PAGE loading buffer, and boiled for western blot analysis on Ubiquitin (Proteintech, 10201-2-AP, 1:200).
5
Immunostaining of Cytoskeletal Proteins
6
ROCK1 Protein Quantification in Cell Lysates
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Total protein extraction from in vitro cultured cells was performed using cell lysis buffer (P0013K, Beyotime), and protein concentration was measured using BCA assay. SDS/PAGE (10% gel) was performed to separate proteins. After gel transfer, PVDF membranes were blocked with 5% skimmed milk at room temperature for 1 h, followed by incubation with primary antibodies including rabbit anti-ROCK1 (1:1500, ab45171, Abcam) and rabbit anti-β-actin (1:1500, ab8227, Abcam) overnight at 4°C. Membranes were washed with TBST (0.3% Tween20), followed by incubation with anti-rabbit IgG-HRP secondary antibody (1:1000, MBS435036, MyBioSource) at room temperature for 2 h. After washing with TBST (0.3% Tween20), ECL (Sigma–Aldrich, U.S.A.) method was used to develop signals, and relative expression level of ROCK1 was normalized to endogenous control β-actin using ImageJ V1.6. This experiment was performed in triplicate manner and data were expressed as mean ± S.D.
7
Immunohistochemistry Staining of Tumor Tissue
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Immunohistochemistry staining was conducted as previously reported [44 ]. The collected tumor tissues were fixed in 5% formalin for 24 h, and embedded in OCT compound. Then, tissues were sectioned into 10-μm thickness. After antigen retrieval using microwave oven, the sections were washed twice (5 min/time) incubated in 10% H2O2 for 5 min. After washing twice (5 min/time), the sections were blocked in 5% goat serum. Primary antibodies anti-ROCK1 (1:100, ab45171, Abcam, UK) and anti-ki67 (1:500, ab15580, Abcam) were applied at 4 °C overnight. Then, the sections were incubated with secondary antibody for 2 h. DAB reagent was added to the sections for developing, and the images were captured under an inverted microscope (Olympus BX41, Tokyo, Japan).
8
Quantifying ROCK1 and ROCK2 in Lymph Nodes
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Formalin-fixed, paraffin wax-embedded blocks from each case were selected for immunohistochemical studies using the antibodies against ROCK1 and ROCK2. Hematoxylin and eosin-stained lymph node tissue slides were used for immunohistochemistry. Control tissue sections were made from the lymph node biopsies of the healthy subjects. Sections of 4 µm were cut from paraffin-embedded tissue blocks onto silane-coated slides. Sections were heated to 60 °C for 20 min prior to deparaffinization with xylene solution. Sections were then stained using the Bond Polymer Refine Detection Kit (Bond #DS9800) in an automated slide processing system (Bond-Max, Leica Microsystems, Buffalo Grove, IL, USA). ROCK1 (rabbit monoclonal, EP786Y, ab45171, Abcam, Cambridge, UK) and ROCK2 (rabbit polyclonal, ab71598, Abcam, Cambridge, UK) were used for ROCK1 and ROCK2 immunostaining, respectively. The percentage of cells staining was evaluated and intensity (–, +, ++, or +++) was scored from 0 to 3 [20 (link)].
9
Quantitative Western Blot Analysis
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Total protein was extracted from EWS cell lines with RIPA buffer (Thermo Scientific, Inc.) according to manufacture instructions. Equal amounts of heat-denatured protein samples (50 mg per lane) were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The antibodies included primary rabbit monoclonal anti-ROCK1 (Ab45171; dilution, 1:500; Abcam, Cambridge, MA, USA), primary rabbit monoclonal anti-ROCK2 (Ab125025; dilution, 1:10,000; Abcam) and rabbit monoclonal anti-GAPDH antibody (AbEPR6256; dilution, 1:10,000; Abcam). The immunoblots were developed using goat anti-rabbit secondary antibody (Ab6721; dilution, 1:5,000; Abcam) followed by detection with the ECL Western Blotting Substrate kit (Abcam, Cambridge, UK) and visualized in a ChemiDoc Bioimaging System (Bio-Rad, Hercules, CA, USA). Expression levels were quantified using ImageJ® software and normalized to loading controls.
10
Immunofluorescence Analysis of Uterine Horn
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Uterine horns collected from either miR-1291 antagomir or NC treated mice were frozen in Tissue-Tek® O.C.T. Compound and sectioned into 9 µm pieces for immunofluorescence staining. Non-specific antigens in the sections were blocked using 10% goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd.) for 1 h following antigen retrieval. Antigen retrieval was performed in a pressure cooker using EDTA (1:50 dilution, pH 9.0) for 20 min in boiling water. Sections were then incubated overnight at 4°C with primary antibodies raised against ArhGAP29 (NBP1-05989; 1:50; Novus Biologicals Canada ULC), RhoA (sc179; 1:100; Santa Cruz Biotechnology, Inc.) and ROCK1 (ab45171; 1:100; Abcam). Subsequently, Alexa Fluor 488 goat anti-rabbit IgG antibody (A11008; 1:200; Thermo Fisher Scientific, Inc.) was used as a secondary antibody and incubated with the sections for 1 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 200 ng/ml) for 10 min at room temperature. Images were captured using an Olympus microscope (IX51; Olympus Corporation, Tokyo, Japan).
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