Cofilin 1 protein expression in bladder cancer tissues and corresponding paracancerous tissues was evaluated by IHC with the 3′, 3′-diaminobenzidine (DAB) kit (ZLI-9017, ZSGB-Bio, Beijing, China), which was performed according to the manufacturer's protocol. Cofilin 1 monoclonal antibody (ab42824, 1:200, Abcam, USA) and goat anti-rabbit HRP antibody (ab136817, 1:400, Abcam, USA) were used.
Ab136817
Manufactured by Abcam
Sourced in United States
Ab136817 is a specific antibody product from Abcam. It is a laboratory reagent used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ab136815, by Abcam (2 mentions)
Dab kit, by ZSGB-BIO (1 mentions)
Ab42824, by Abcam (1 mentions)
Ab15200, by Abcam (1 mentions)
S21024 2, by Thermo Fisher Scientific (1 mentions)
Ad0023 100, by Sangon (1 mentions)
A5228, by Abcam (1 mentions)
Sc 11769, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ab119557, by Abcam (1 mentions)
Ab6785, by Abcam (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Evans blue, by Merck Group (1 mentions)
Apocynin, by Merck Group (1 mentions)
Ab129068, by Abcam (1 mentions)
Ab33186, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Ab45171, by Abcam (1 mentions)
Anti claudin 5, by Santa Cruz Biotechnology (1 mentions)
Ab179471, by Abcam (1 mentions)
9 protocols using ab136817
1
Cofilin 1 Expression in Bladder Cancer
2
Histopathological Evaluation of Liver Tissue
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The formalin fixed and paraffin-embedded liver tissue slices (5 μm thickness) were deparaffinized and rehydrated for histopathological evaluation [21 (link),22 (link)]. For sirius red staining, the sections were stained by hematoxylin and sirius red. For immunohistochemical staining, the sections were incubated with 0.3% hydrogen peroxide/phosphate-buffered saline for 30 min and blocked with 10% BSA (Sangon, AD0023-100). Slides were first incubated using the antibody for CTHRC1 (Huabio), α-SMA (Sigma, A5228) or desmin (Abcam, ab15200) at 4 °C overnight with optimal dilution, labeled by HRP second antibody of mouse (Cell Signaling, 5470S) or rabbit (Abcam, ab136817) at room temperature for 1 h. Then the sections were treated with DAB substrate liquid (Thermo, S21024-2) and counterstained by hematoxylin. All the sections were observed and photographed with a microscope (Carl Zeiss).
3
Protein Expression and Signaling Analysis
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Immunohistochemistry and immunocytochemistry were performed to examine the expression of ESRP, E-cadherin, and vimentin in cells according to the manufacturer’s instructions. Western blotting assays were also performed using whole cell lysates. Cell lysates were prepared and separated by SDS/PAGE. Proteins were transferred on to PVDF membrane, and membranes probed primary antibodies including ESRP1/2 (anti-RBM35A+RBM35B, ab106555, Abcam), Smad (ab207447, Abcam), or p-Smad (sc-11769, Santa Cruz Biotechnology) overnight at 4°C, then blotted with corresponding secondary antibodies (ab136817 and ab6785, Abcam). Visualization was accomplished using BioRad ChemiDoc™ XRS system (Hercules, CA) with electrochemiluminescence substrate. Protein levels were normalized to match densitometric values of internal controls. In addition, ELISA was performed to examine the level of TGF-β1 (ab119557, Abcam) in BALF according to the manufacturer’s instructions.
4
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).
5
Protein Analysis via Western Blotting
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Cells were lysed in RIPA buffer and lysates were sonicated and then boiled and centrifuged (447.2 × g). Proteins were resolved with 8–15% SDS-PAGE and transferred to PVDF membranes. After blocking, membranes were incubated with the indicated primary antibodies and then horseradish peroxidase-conjugated secondary antibodies (1:4,000; ab136817; Abcam). Blots were visualized using ECL and SuperSignal reagent (WBKLS0500; EMD Millipore, Billerica, MA, USA). Protein quantification was conducted using image J software.
6
Protein Expression Analysis in Testicular Tissue
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Total protein was extracted from testicular tissue. Western blot analysis was conducted as previously published by our laboratory [22 (link)]. The membrane was incubated with Anti-Integrin α6 (1 : 2000 dilution; ab181551, Abcam), Anti-Integrin β1 (1 : 2000 dilution; ab179471, Abcam), phospho-Akt polyclonal rabbit antibody at a dilution of 1 : 2000 (ab81283, Abcam), and phosphoinositide 3-kinase (PI3K) at a dilution of 1 : 2000 (4257, CST) overnight at 4°C and incubated with anti-rabbit IgG horseradish peroxidase conjugated secondary antibodies (1 : 3000 dilution; ab136817, Abcam). The relative protein expression was normalized with GAPDH (1 : 3000 dilution; ab8245, Abcam).
7
Western Blot Analysis of Signaling Pathways
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Total protein extracts from the cultured foam cell lines and thoracic aorta tissues were prepared in direct lysis buffer [50 mmol/l Tris-HCl (pH 6.8), 100 mmol/l DTT, 2% SDS, 10% glycerol, 1X complete (Roche, Mannheim, Germany) and 0.2% bromophenol blue]. The cells were boiled for 10 min and centrifuged at 13,000 rpm for 5 min. Following 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were electroblotted onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). The blots were then probed with primary antibodies against p38 (1:5,000), p-p38 (1:1,000), ERK (1:2,000), p-ERK (1:1,000), JNK (1:2,500), p-JNK (1:200), p65 (1:5,000) and PDCD4(1:5000) followed by incubation with the appropriate secondary antibodies, including goat anti-mouse IgG-H&L (HRP) (Abcam; ab136815; 1:10,000) and goat anti-rabbit IgG-H&L (HRP) (Abcam; ab136817; 1:10,000). Protein signals were visualized by using the enhanced chemiluminence (ECL) Western blot detection system (Millipore, Billerica, MA, USA) and quantified by densitometry. All the figures illustrating western blot analyses are representative of at least 3 independent experiments.
8
Western Blot Protein Analysis
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Cells were collected and washed three times with PBS before lysis in lysis buffer (MK163780, ThermoFisher Scientific Inc, Waltham, MA, USA). The protein concentration of the lysates was determined according to the BCA method using the Protein Quantitative Analysis kit (2812 k; CWBIO, CoWin Biotech Co. Ltd., Beijing, China). For electrophoresis, 100 μg of protein was loaded into the wells of a 10% SDS polyacrylamide gel. After separation, proteins were transferred to nitrocellulose membranes, blocked for 3 h at room temperature in blocking buffer (5% nonfat dry milk, Tween-Tris-buffered saline), washed in PBS 3 times and incubated with primary antibody of ABCAM (Rabbit polyclonal to ERK, ab16869, 1: 1:5000; Rabbit polyclonal to AKT, ab66138, 0.15 μg/ml; Rabbit polyclonal to Bax, ab7977, 1:1000; Rabbit polyclonal to Bcl-2, ab18210, 1 μg/ml) and Actin (sc-1616, 1:200, SANTA CRUZ BIOTECHNOLOGY) overnight at 4°C with gentle agitation. Next day after washing three times in PBS, the nitrocellulose membranes were blotted for 1 h at room temperature with secondary antibodies (ab136817, 1:5000, abcam). Finally, immunolabeled protein bands were detected using the ECL(Electro-Chemi-Luminescence) method (Immobilon™ Western, Millipore Corporation, Billerica, MA, USA) and quantified using an Alpha Imager 2200 (ProteinSimple, Santa Clara, CA, USA).
9
Western Blot Analysis of Cellular Proteins
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Western blot analysis was conducted as previously published by our laboratory 21 (link). Briefly, 40 µg protein samples were electrophorsed on a 10% SDS-PAGE gel, transferred to PVDF membrane (Millipore, Billerica, MA, USA), and then probed with primary antibodies against LBH (mAb, Sigma), Cyclin D1 (mAbs, CST), cyclin E2 (mAbs, CST) and GAPDH (mAbs, CST). A horseradish peroxidase-conjugate anti-mouse and anti-rabbit IgG (1:3000; ab136815, ab136817, Abcam, UK) was then added, and the secondary antibody was detected through autoradiography using enhanced chemiluminescence (ECL Plus general electric healthcare Milwaukee, WI).
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