Western blot analysis was performed as previously reported (Liu et al., 2015 (link); Li et al., 2015 (link)). Three mice from each group were sacrificed after WMW test, and the hippocampal and cortical tissues were collected and homogenized in RIPA lysis buffer (1:5, w/v). Protein concentrations were determined by BCA protein assay kit. An aliquot of 45 µg of protein was applied for electrophoresis followed by the transferring of protein to the polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk in TBST buffer for one hour at room temperature, and incubated with the primary antibody: anti-CNTF (1:1,000), BDNF (1:1,000), and GDNF (1:1,000) at 4 °C overnight. Next, HRP-labeled goat anti-rabbit IgG (Beyotime Biotechnology, Jiangsu, China; A0208, 1:2,000) was incubated with the membrane at room temperature for one hour. The blots were visualized using the enhanced ECL Western blot detection kit (7Sea Biotech, Shanghai, China) and scanned to Gel Imaging. The band intensity was quantified using Quantity One 1-D analysis software v4.52 (BioRad, Hercules, California, USA).
Quantity one 1 d analysis software v4
Manufactured by Bio-Rad
Sourced in United States
Quantity One 1-D analysis software v4.52 is a comprehensive software solution for analyzing 1-dimensional gel electrophoresis data. It provides tools for image capture, band/spot detection, quantification, and presentation of results.
Automatically generated - may contain errors
Lab products found in correlation
Molecular imager gs 800, by Bio-Rad (2 mentions)
Hrp labeled goat anti rabbit igg, by Beyotime (1 mentions)
Horseradish peroxidase labeled goat anti rabbit igg, by Beyotime (1 mentions)
α synuclein, by Abcam (1 mentions)
Lc3 1 2, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Sqsttm1 p62, by Cell Signaling Technology (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
β actin, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Chemiluminescence detection system, by Cytiva (1 mentions)
Proteome profiler mouse phospho rtk array kit, by R&D Systems (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
14 protocols using quantity one 1 d analysis software v4
1
Protein Expression Analysis in Hippocampus and Cortex
2
Hippocampal Protein Expression Analysis
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Five mice from each group were euthanized after the MWM test, and the hippocampal tissues of half of the brains were collected and homogenized in radioimmunoprecipitation assay lysis buffer (1:5, w/v). Protein concentrations were determined by a bicinchoninic acid protein assay. A total of 45 μg of protein was applied. After electrophoresis, the bands were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in tris-buffered saline with Tween 20 buffer for 1 h at 22 ± 2°C, then incubated with a primary antibody: anti-Aβ1-40 (1:1000), anti-Aβ1-42 (1:1000), LC3A/B (1:1000), beclin1 (1:1000), p70S6 kinase (p70S6K) (1:1000), Pp70s6k (1:1000), cat D (1:1000), cat B (1:1000), or v-ATP (1:500) at 4°C overnight. The following day, after incubation with horseradish peroxidase-labeled goat anti-rabbit IgG (Beyotime Biotechnology, China; A0208, 1:2000) or goat anti-mouse IgG (1:5000) at 22 ± 2°C for 1 h. The blots were visualized using an enhanced chemiluminescence western blot detection kit (7Sea Biotech, China) and scanned with the Gel Imaging system. The band intensity was quantified using Quantity One 1-D analysis software v4.52 (BioRad, Hercules, CA, United States).
3
Quantitative Protein Analysis of Neuronal APP Pathways
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Total protein was extracted from cultured neurons using a total protein extraction kit and quantified by a BCA protein assay kit. Equal amounts of protein (20 µg) per lane were separated by SDS-PAGE gels and then transferred to a PVDF (0.45 µm) membrane. The membranes were incubated with the following primary antibodies: anti-APP (1:500), anti-ADAM10 (1:1,000), anti-ADAM17 (1:1,000), anti-BACE1 (1:1,000), anti-PS1 (1:1,000), anti-Aβ1–42 (1:1,000), anti-β-actin (1:2,000), and anti-GAPDH (1:2,000) at 4 °C overnight, followed by incubation with secondary antibody at 4 °C for 1 h. The membranes were visualized using the chemiluminescence reagent ECL Plus (E003-100). The image was scanned, and band densities were quantified using Quantity One 1D analysis software v4.52 (Bio-Rad). GAPDH or β-actin was used to normalize protein loading.
4
Protein Expression Analysis in Neuronal Tissues
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The protein expressions in SN tissues or PC12 cells were analyzed by Western blot analysis. The brain was removed under anesthesia. Each brain was dissected on a cold glass plate to separate the SN tissues. The SN tissues or PC12 cells pellets were homogenized in RIPA lysis buffer. The lysates were incubated on ice for 30 min, then centrifuged at 12,000 g for 15 min. The supernatant was then collected for the analysis of protein expressions. Determination of protein concentrations was carried out by BCA kit (Beyotime, Beijing, China). The protein was separated on 10% Bis-Tris NuPAGE gel and transferred to PVDF membrane. The PVDF membrane was blocked with 4% BSA (Sigma) for 2 h, and then reacted with primary antibodies at 4℃ for overnight. The primary antibodies included those for α-synuclein (1:1000, Abcam), LC3-I/II (1:1000, Abcam), Beclin-1 (1:1000, Cell signaling), SQSTTM1/P6
2 (1:1000, Cell signaling), mTOR (1:1000, Cell signaling), Phospho-mTOR (1:1000, Cell signaling), and β-actin (1:2000, Beyotime). After washing, the membranes were then incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 2 h. The membrane-bound secondary antibody was detected with ECL Western blot detection kit. The band intensities were quantified using Quantity One 1-D analysis software v4.52 (BioRad).
2 (1:1000, Cell signaling), mTOR (1:1000, Cell signaling), Phospho-mTOR (1:1000, Cell signaling), and β-actin (1:2000, Beyotime). After washing, the membranes were then incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 2 h. The membrane-bound secondary antibody was detected with ECL Western blot detection kit. The band intensities were quantified using Quantity One 1-D analysis software v4.52 (BioRad).
5
Oxidative Stress-Induced Autophagy in PC12 Cells
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PC12 cells were pre‐treated with different concentrations of ICS II for 1 hr before exposure to 400 μM H2O2. After 48 hrs, cells were lysed in the lysis buffer, and proteins were quantified using the BCA protein assay kit. Following electrophoresis, proteins were blotted onto a PVDF membrane. The membrane was blocked with 5% non‐fat milk at room temperature for 2 hrs and incubated overnight at 4°C with the appropriate primary antibody: anti‐LC3B (1:1000), anti‐Beclin 1 (1:1000), anti‐p62/SQSTM1 (1:1000), anti‐HSP60 (1:1000), anti‐GSK‐3β (1:1000), anti‐p‐ser9‐GSK‐3β (1:1000), anti‐p‐tyr216‐GSK‐3β (1:1000). Thereafter, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies for 2 hrs at room temperature under shaking. Immunoreactive proteins were visualized with the ECL Western blot detection kit. The image was scanned and band optical intensity was quantified using Quantity One 1‐D analysis software v4.52 (BioRad, Philadelphia, USA).
6
Western Blot Analysis of Autophagy and Signaling Pathways
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Protein lysates were prepared from cultured SW1353 cells using a protein extraction kit, and the concentrations of sample lysates were determined using the BCA assay. After completion of SDS–PAGE electrophoresis, separated proteins in the gel were transferred onto a PVDF membrane. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated overnight at 4℃ with the following primary antibodies: collagen II, MMP-3, LC3, p62, Beclin1, ULK1, PI3K, AKT, mTOR, p-PI3K, p-AKT, p-mTOR, and GAPDH (as the internal control). On the next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (according to the animal species of the primary antibodies) for 1 h at room temperature with shaking. The protein bands were detected with the ECL Western blot reagent. The blots were visualized using the Davinch-Chemi™ imaging system. Quantity One 1-D analysis software v4.52 (BioRad, Philadelphia, USA) was used to quantify the relative optical intensity of the protein bands.
7
Quantification of Apoptosis Markers in Cultured Neurons
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Total protein was extracted from cultured neurons using a total protein extraction kit and quantified by a BCA protein assay kit. Equal amounts of protein (20 mg) per lane were separated by Nu-PAGE gels and then transferred to a PVDF (0.45 mm) membrane. The membranes were incubated with the following primary antibodies: anti-Bax (1 : 1000), anti-Bcl2 (1 : 1000), anti-caspase-9 (1 : 1000), anti-caspase-3 (1 : 1000), anti-GAPDH (1 : 2000), and anti-β-actin (1 : 2000) at 4°C overnight, followed by incubation with secondary antibody at 4°C for 1 h. The membranes were visualized using the chemiluminescence reagent ECL Plus (E003-100). The image was scanned, and band densities were quantified using Quantity One 1D analysis software v4.52 (Bio-Rad).
8
Quantitative Analysis of Gene and Protein Expression
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Total RNA was extracted from cells and tissues using Trizol (Life technologies, NY) according to the manufacturer's instructions. Upon isolating RNA, DNase I was treated to eliminate contaminating genomic DNA. RevertAid First Strand cDNA Synthesis Kit (Life technologies, NY) was used for cDNA synthesis. All results were quantified by qPCR performed using GoTaq qPCR Master Mix (Promega, WI) on a BioRad CFX96 (Bio-Rad, CA). Gene expression levels were analyzed using the delta Ct method and normalized against β-actin. The gene-specific primers used in RT-qPCR were listed in Table 1 .
For human tissues, samples were harvested and lysed in RIPA buffer using a dounce homogenizer, followed by sonication and incubation on ice for 15 min. Pellets were spun down and the supernatant was collected. For cells, whole lysates were prepared by direct lysis in RIPA buffer. Proteins were quantified using Pierce BCA Protein Assay Kit (Life technologies, NY) and 40 μg per well were loaded. Samples were then separated by SDS-PAGE electrophoresis and transferred to nitrocellulose for detection using antibodies.
The abundance of the proteins in western blot assays was determined by densitometry using Quantity One 1D Analysis Software v4.6 (Bio-Rad, CA).
For human tissues, samples were harvested and lysed in RIPA buffer using a dounce homogenizer, followed by sonication and incubation on ice for 15 min. Pellets were spun down and the supernatant was collected. For cells, whole lysates were prepared by direct lysis in RIPA buffer. Proteins were quantified using Pierce BCA Protein Assay Kit (Life technologies, NY) and 40 μg per well were loaded. Samples were then separated by SDS-PAGE electrophoresis and transferred to nitrocellulose for detection using antibodies.
The abundance of the proteins in western blot assays was determined by densitometry using Quantity One 1D Analysis Software v4.6 (Bio-Rad, CA).
9
Phospho-RTK Array Profiling of DRG
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Proteome profiler mouse phospho-RTK array kit (R&D system, ARY014) was used to quantify the phosphorylation level of 39 tyrosine kinase receptors, following the manufacturer’s instructions. Briefly, the membrane containing immobilised phospho-RTK was initially blocked with BSA at room temperature for 1 h, followed by an overnight incubation with the lysates of embryonic and adult DRG from retrieved and in vitro conditions, at 4°C. After a series of washes, the membrane was incubated with a Detection Antibody Cocktail, then with horseradish peroxidase-conjugated secondary antibody (Pierce Chemical, Rockford, IL) and finally with a chemiluminescent detection reagent. Each array membrane was exposed to X-ray film using a chemiluminescence detection system (Amersham). The film was scanned using Molecular Imager GS800 calibrated densitometer (Bio-Rad) and pixel density was presented by quantifying the mean spot densities with Quantity One 1-D Analysis Software, v 4.6 (Bio-Rad).
10
Phospho-RTK Array Analysis of DRG
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A proteome profiler mouse phospho-RTK array kit (#ARY014, R&D system, USA) was used to quantify the phosphorylation level of 39 RTKs. After 72 h of treatment with conditioned medium and controls, the protein lysate of DRG was quantified and analyzed. According to the manufacturer’s instructions, for the array analysis, the same amount of protein was added to each membrane (100 µg). Each array membrane was exposed to X-ray film using a chemiluminescence detection system (Amersham, GE Healthcare). The film was scanned using Molecular Imager GS800 calibrated densitometer (Bio-Rad, Hercules, USA), and pixel density was quantified using Quantity One 1-D Analysis Software, v 4.6 (Bio-Rad). The results were presented as the mean spot intensity, which corresponds to the mean of the two spots for each receptor within the same membrane array.
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