SKCs were transfected with oar-miR-200c mimics (or inhibitor) and collected 48 h later. After treating with protein lysate (RIPA：PMSF=100：1), the supernatant was collected by centrifugation at 12,000 g for 5 min at 4℃. Total protein content was determined by a BCA protein assay kit (Beyotime, China). Approximately 30 μg of extracted protein with loading buffer was loaded into wells of a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for electrophoresis and then transferred to a nitrocellulose (NC) membrane (Millipore, USA). After blocking, the membranes were treated with anti-ZEB1 (1：3,000, Abcam), anti-β-actin (1：5,000, Abcam), and anti-E-cadherin (1：2,500, BD Biosciences) antibodies and incubated overnight at 4℃. Then, the membranes were treated with horseradish peroxidase-conjugated secondary antibody (Bioworld, China) for 1 h at 37℃. The protein bands were visualized using a gel imager (Bio-Rad, USA).
Horseradish peroxidase conjugated secondary antibody
Manufactured by Bioworld Technology
Sourced in United States, China
Horseradish peroxidase-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of a secondary antibody that is chemically conjugated to the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of a specific target antigen or protein in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
P iκbα, by Cell Signaling Technology (2 mentions)
β actin, by Bioworld Technology (2 mentions)
Nitrocellulose nc membrane, by Merck Group (1 mentions)
Anti e cadherin, by BD (1 mentions)
Gel imager, by Bio-Rad (1 mentions)
Anti β actin, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti zeb1, by Abcam (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Mmp 9, by Abcam (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Immobilon p polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Gapdh antibody, by Merck Group (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Anti ccl5, by Abcam (1 mentions)
Anti il 1β, by Merck Group (1 mentions)
18 protocols using horseradish peroxidase conjugated secondary antibody
1
Analyzing ZEB1, E-cadherin Expression in miR-200c Transfected Cells
2
Western Blot Analysis of Signaling Proteins
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Western blotting was performed as reported previously.13 The polyvinylidene fluoride membranes were first incubated with primary antibodies MMP‐9 (1:1000; Abcam), pERK, ERK, pIκBα, IκBα (1:1000; Cell Signaling Technology), and glyceraldehyde 3‐phosphate dehydrogenase (1:3000; PeproTech) overnight at 4°C separately, and then incubated with horseradish peroxidase‐conjugated secondary antibody (Bioworld). Color development was achieved by ECL reagents. More information can be found in Supporting Information Material.
3
Western Blot Analysis of Protein Expression
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Total proteins from cells were harvested and lysed in radioimmunoprecipitation buffer supplemented with 1% protease inhibitors (Pierce, Dorchester, MA, USA, catalog number: 88668). Equal amounts of the total protein were separated on 7.5%–10% SDS-polyacrylamide gel and transferred to 0.45 μm PVDF membranes [38 (link)] (Millipore, Burlington, MA, USA, catalog number: IPVH00010). After being blocked with 5% skimmed milk for 1 h, the membranes were incubated with the primary antibodies for GAPDH (1:5000, Bioworld, Minneapolis, MN, USA, catalog number: AP0063), Vimentin, SOX2 (1:1000, Bioworld, Minneapolis, MN, USA, catalog number: BS1491, MB0064), E-cadherin, N-cadherin (1:1000, CST, USA, catalog number: 3195T, 13116P), NANOG, LIN28 and CD44 (1:500, SAB, Los Angeles, CA, USA, catalog number: 21423, 21626) at 4 °C overnight. After being washed three times with tris-buffered saline and Tween, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, Bioworld, Minneapolis, MN, USA) for 1 h. Then, the protein bands were visualized using an enhanced chemiluminescent substrate detection system (Millipore, Burlington, MA, USA, catalog number: WBKLS0500).
4
Western Blot Analysis of Alkaline Phosphatase
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To assess alkaline phosphatase (ALP) protein expression, we performed Western blot
analysis, as described elsewhere (30 (link)). In
brief, proteins were extracted from different experimental groups at day 10 and
quantified. Twenty micrograms of supernatant protein samples were subjected to sodium
dodecylsulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P
polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Following blocking,
immunoblots were incubated with anti-ALP monoclonal antibody (1:10,000; Abcam, UK)
overnight at 4°C. A GAPDH antibody (Sigma) was used as a protein loading control.
Blots were then incubated with horseradish peroxidase-conjugated secondary antibody
(1:10,000; Bioworld, USA) at 37°C for 1 h and visualized using a SuperSignal West
Pico chemiluminescence substrate kit (Pierce, USA). The membranes were scanned using
a Molecular Imager (Bio-Rad, USA), followed by data analysis using the Image Lab
software (Bio-Rad). Data are reported as the protein-to-GAPDH ratio to correct for
variations in protein loading.
analysis, as described elsewhere (30 (link)). In
brief, proteins were extracted from different experimental groups at day 10 and
quantified. Twenty micrograms of supernatant protein samples were subjected to sodium
dodecylsulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P
polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Following blocking,
immunoblots were incubated with anti-ALP monoclonal antibody (1:10,000; Abcam, UK)
overnight at 4°C. A GAPDH antibody (Sigma) was used as a protein loading control.
Blots were then incubated with horseradish peroxidase-conjugated secondary antibody
(1:10,000; Bioworld, USA) at 37°C for 1 h and visualized using a SuperSignal West
Pico chemiluminescence substrate kit (Pierce, USA). The membranes were scanned using
a Molecular Imager (Bio-Rad, USA), followed by data analysis using the Image Lab
software (Bio-Rad). Data are reported as the protein-to-GAPDH ratio to correct for
variations in protein loading.
5
Quantifying Cellular Protein Levels
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Proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer. A total of 30 µg of protein was subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and blotting onto a polyvinyl difluoride membrane. After blocking, the membrane was incubated with anti-CCL5 (Abcam) or anti-GAPDH antibody (Abcam) overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary antibody (Bioworld, Nanjing, Jiangsu, China) for 2 hours. The target band was detected using an ECL detection reagent (Pierce/Thermo Fisher Scientific).
6
Western Blot Analysis of Cell Cycle Proteins
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HELF cells were lysed in cold lysis buffer [40 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol and 1 mM fresh phenylmethylsulfonyl fluoride]. Lysates were centrifuged at 12,000 × g for 15 min. The supernatant was collected and the protein concentration was determined by Lowry protein assay. Next, 20 μg protein was loaded in each well, electrophoresed by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk in PBS with 0.5 ml/l Tween-20 for 2 h at room temperature, and incubated overnight at 4°C with primary antibodies against human P16, P27, P53 or Rb proteins, purchased from Beyotime (Nanjing, China). This was followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Bioworld Technology, St. Louis Park, MN, USA) at room temperature for 1 h. Enhanced chemiluminescence was used to detect the results.
7
Western Blot Analysis of Protein Markers
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The protein lysates were prepared with protease inhibitors cocktail‐contained radioimmunoprecipitation assay lysate (Beyotime). The protein samples were boiled at 98°C for 10 min. Next, 60 μg of proteins was used for analysis on 10% sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (SDS‐PAGE; Bio‐Rad, Hercules, CA). The SDS‐PAGE gels were transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes (Bio‐Rad) for 45 min. Then, the protein‐carried PVDF membranes were blocked with 2% bovine serum albumin, and they were incubated with the indicated primary antibody overnight. Afterward, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody for 45 min diluted at 1:50,000 (Bioworld Technology, Nanjing, China). Importantly, the primary antibodies including anti‐GAPDH, anti‐FBXW7, anti‐Bax, anti‐Bcl‐2, anti‐TNF‐α, anti‐IL‐1β, anti‐IL‐6, anti‐CAT, anti‐GSH, and anti‐SOD1 were purchased from Sigma‐Aldrich.
8
Analyzing MRP Complex Expression in E. coli
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Western blotting was used to determine the expression of Mrp complex’s subunits in E. coli KNabc. Briefly, cells grown in LBK medium were harvested and resuspended in buffer A and subjected to homogenization using a JY92-IIN Ultrasonic homogenizer. The membrane fraction was collected by ultracentrifugation at 100,000 × g for 30 min. The pellet membrane fraction was solubilized in buffer A supplemented with 1% (wt/vol) DDM for 1 h at 4 °C. After second ultracentrifugation at 100,000 × g for 30 min, the supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 15% polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane at 20 V for 16 min using Semi Dry Transfer. Immunoblotting was performed using anti-strep II antibody (Bioworld Technology) with 1:5,000-fold dilution, horseradish peroxidase-conjugated secondary antibody with 1:10,000-fold dilution (Bioworld Technology).
9
Sciatic Nerve Injury Proteome Analysis
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The lesioned sciatic nerve tissues were lysed in a Laemmli sample buffer (2% SDS, 52.5 mM Tris-HCl PH 6.8, and protein inhibitors). The concentration of protein lysates was determined using the Micro BCA Protein Assay Kit (Beycotime Biotechnology, P0010). Eighty micrograms of proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA). Afterwards, the membranes were blocked with 5% nonfat milk and were probed with primary antibodies including: ATG-7 (1:1000), ATG-5 (1:1000), P62 (1:1000), Beclin-1 (1:1000), LC3 (1:1000), p-AMPK (1:500), AMPK (1:1000), p-p70s6k (1:500), p70s6k (1:1000), p-mTOR (1:500), mTOR (1:1000), MBP (1:1000), and MPZ (1:1000) overnight at 4°C. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000; Bioworld; anti-rabbit, BS13278; anti-mouse, BS50350) for 1 h. Immunoreactive bands were visualized using the ChemiDic TM XRS + Imaging System (Bio-Rad, 1708195). Densitometric quantification of the membranes was obtained using Image J software (National Institutes of Health, USA). Experiments were repeated three times and GAPDH (1:10,000) or β-actin (1:5000) was used as an internal control.
10
Quantitative Protein Expression Analysis
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HEK293T cells were lysed in radioimmunoprecipitation buffer (EpiZyme, PC103) with 1 mM PMSF solution and a mixture of protease inhibitors (Roche), at about 30 h after transfection. Lysates were kept on ice for 30 min, followed by centrifugation for 30 min at 12,000 rpm at 4 °C. Then, the supernatants were mixed with 5× loading buffer (Mei5bio, MF145-01) before being separated by 6% SDS-PAGE gels (Beyotime, P0050A). Proteins were immunoblotted onto polyvinylidene difluoride membranes (Millipore), which were blocked in 5% nonfat milk dissolved in 150 mM NaCl, 10 mM Tris-Base (pH 7.4), and 0.1% Tween 20 at RT for 1 h. The membranes were then incubated with anti-HA (Cell signaling technology, H3724, 1:1000), anti-FLAG (Sigma–Aldrich, F7425, 1:1000), anti-TMEM63B (abcam, 1:1000), anti-β-COP (abcam, ab2899, 1:1000), anti-Gapdh (Bioworld, MB001, 1:10,000) primary antibodies, and horseradish peroxidase–conjugated secondary antibodies (Bioworld). The reactive protein bands were developed by ECL kit (Tanon, 180–501) and captured by a chemiluminescent imaging system (Tanon). To quantify protein expression, the integrated absorbances of protein bands were measured using ImageJ software (National Institutes of Health).
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