The cortical tissue (30 mg) surrounding the lesioned areas were lysed in lysis buffer (Santa Cruz Biotechnology), and the total protein levels were quantified. A total of 25 μg protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane that were blocked in 10% skimmed milk at room temperature for 2 h. The solution was incubated at 4°C overnight with primary antibodies against H3, H4, acetyl-H3, acetyl-H4, acetyl-Nrf2 (Cell Signaling Technology), cleaved caspase-3, LC3, Beclin, ATG-3 and ATG-7 (Cell Signaling Technology), Bax, ROS and GFAP (Abcam), Iba-1, Nrf2, HO-1, NQO1 and UGT1A1 (Santa Cruz Biotechnology Inc.), followed by incubation with appropriate secondary antibodies. Immunoblots were visualized using the Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Gray value analysis was conducted with the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or Lamin B1 (1:3000, Cell Signaling Technology).
Un scan it 6
Manufactured by Silk Scientific
Sourced in United States
The UN-Scan-It 6.1 software is a data digitization tool designed for accurately extracting numerical data from graphs, charts, and other visual representations. It allows users to quickly and efficiently convert analog data into digital formats for further analysis and processing.
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Lab products found in correlation
Ecl western blotting detection system, by Merck Group (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Sirt1, by Santa Cruz Biotechnology (3 mentions)
β actin, by Bioworld Technology (3 mentions)
Lamin b1, by Cell Signaling Technology (2 mentions)
Laminin b1, by Cell Signaling Technology (2 mentions)
Bcl 2, by Abcam (2 mentions)
Histone h3, by Bioworld Technology (2 mentions)
Enhanced chemiluminescence solution, by Thermo Fisher Scientific (2 mentions)
Ac foxo1, by Santa Cruz Biotechnology (2 mentions)
Ac p53, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Bcl 2 associated x factor bax, by Abcam (2 mentions)
Iba 1, by Cell Signaling Technology (2 mentions)
Cd206, by Abcam (2 mentions)
Nf κb p65, by Cell Signaling Technology (2 mentions)
Lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Millipore ecl western blotting detection system, by Merck Group (1 mentions)
12 protocols using un scan it 6
1
Western Blot Analysis of Protein Expression in Cortical Tissue
2
Protein Extraction and Western Blot Analysis
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Cells were lysed in SDS lysis buffer (0.1 M TRIS pH 7.5, 10% SDS, 1 mM NaVO3, and Complete protease inhibitor mix (Roche)) heated to 90 °C. Protein concentrations were determined using DC assay (BioRad, Hercules, CA). Samples were mixed with sample loading buffer (Life Technologies). Samples and BenchMark ladder (Life Technologies) were separated in BioRad Tris/Glycine/SDS running buffer on pre-made 10% Bis-Tris gels (BioRad). Proteins were transferred to PVDF membranes using Trans-Blot Turbo transfer system (BioRad), Ponceau S stained, and blocked for 1 h at 37 °C in 5% non-fat dry milk in TBST (0.01 M Tris/HCl, 0.15 M NaCl, 0.1% Tween 20, pH 7.4). Densitometric analyses were carried out using UN-SCAN-IT 6.1 software (Silk Scientific, Orem, Utah).
3
Protein Expression Analysis in Cells
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Proteins were extracted with radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology). Proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane that was probed with primary antibodies against B cell lymphoma (Bcl)-2 (1:400), Bax (1:200), LC3-1(1:400) and P62 (1:400), (all from Abcam); HO-1 (1:200), NQO1 (1:200), and UGT1A1 (1:200) (all from Santa Cruz Biotechnology Inc); and cleaved caspase-3 (1:200), Beclin-1 (1:200), ATG-3 (1:400), and ATG-7 (1:400; all from Cell Signaling Technology), followed by incubation with appropriate secondary antibodies. Immunoreactivity was visualized with the ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Gray value analysis was conducted with the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or laminin B1 (1:3000, Cell Signaling Technology).
4
Protein Extraction and Western Blot Analysis
5
Immunoblotting for Protein Expression
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Cells were harvested with Versene (8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na2PO4, 0.2 g/l EDTA, 0.1 g/l Phenol red, pH 7.34), washed in ice-cold PBS and lysed on ice in a RIPA buffer with fresh protease inhibitors (Roche Complete, Roche Applied Science, Basel, Switzerland) and Phosphatase inhibitors (PhosSTOP, Roche Applied Science). Protein (25 μg) was electrophoresed through a 4–12% Bis-Tris gel, blotted to a PVDF membrane (iBLOT, Invitrogen) and immunoblotted using antibodies to: β-tubulin (Sigma T8328, Sigma-Aldrich, St. Louis, MO, USA), Mcl1 (Santa Cruz SC-819, Santa Cruz Biotechnology, Dallas, TX, USA), PSMD14 (Sigma HPA002114, Sigma-Aldrich), PRPF8 (Abcam ab79237, Cambridge, MA, USA), and SART1 (Abcam ab88583). Rabbit anti-Hub1(UBL5) antibody was a kind gift of Dr. Hideki Yashiroda (University of Tokyo, Tokyo, Japan). Blots were scanned, and densitometry was performed by the UN-SCAN-IT 6.1 software (Silk Scientific Inc, Orem, UT, USA).
6
Western Blot Analysis of Protein Targets
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First, equal amounts of protein were leaded in each lane of a polyacrylamide gel. Next, the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C. The membrane was then washed and incubated for 2 h at room temperature with horseradish peroxidase-conjugated IgG. Finally, we employed ECL Western blotting detection reagents (Thermo Fisher Scientific) to visualize the protein bands and used Un-Scan-It 6.1 software (Silk Scientific, Orem, UT, USA) to measure the band density.
7
Protein Expression Analysis in Neuroinflammation
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Proteins were extracted with radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology). Proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane that was probed with primary antibodies against B cell lymphoma (Bcl)-2 (1:400), Bcl-2-associated X factor (Bax) (1:200), CD16 (1:200), CD206 (1:200); and GFAP (1:400) (all from Abcam); and cleaved caspase-3 (1:200), Iba-1 (1:100), and NF-κB p65 (1:200) (all from Cell Signaling Technology), followed by incubation with appropriate secondary antibodies. Immunoreactivity was visualized with the ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Gray value analysis was conducted with the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or laminin B1 (1:3000, Cell Signaling Technology).
8
Western Blot Analysis of Protein Expression
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Proteins were extracted with a radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology). The BCA method (KeyGEN Biotech, KGPBCA) was used for the protein quantitation. A total of 25 μg protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane by wet transfer. The membrane was then blocked in 10% skim milk at room temperature for 2 h and then incubated at 4 °C overnight with primary antibodies against the following proteins: B cell lymphoma (Bcl)-2 (1:400), Bcl-2-associated X factor (Bax) (1:200), CD16 (1:200), and CD206 (1:200) (all from Abcam), and cleaved caspase-3 (1:200), Iba-1 (1:100), and NF-κB p65 (1:200) primary antibodies (all from Cell Signaling Technology), followed by incubation with the appropriate secondary antibodies (goat anti-rabbit IgG- HRP or goat anti-mouse IgG-HRP, 1:3500) at room temperature for 1 h. Immunoreactivity was visualized with the ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). A gray value analysis was conducted with the UN-Scan-It 61 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or Lamin B1 (1:3000, Cell Signaling Technology).
9
Protein Expression Analysis Protocol
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As previously described, diverse proteins from the specimens were extracted. The Bradford method was applied for protein concentrations. We used 10% or 12% SDS-PAGE to separate the proteins into different bands. Then the separated proteins were transferred onto polyvinylidene-difluoride films. Films were blocked with 5% milk–Tris-buffered saline/0.05% Tween 20 (TBST) for 2 hours. Next, they were incubated at 4°C overnight using primary antibodies of rabbit anti-Nrf2 (1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-HO-1 (1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-NQO1 (1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-cleaved caspase-3 (1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-GAPDH (1:5,000; Bioworld Technology, St Louis Park, MN, USA), and rabbit anti-Histone 3 (1:1,000, Cell Signaling Technology, Beverly, MA, USA). After being washed in TBST, films were incubated with the appropriate secondary antibodies for 1 hour at room temperature. Finally, proteins were examined using an enhanced chemiluminescence (ECL) kit (Millipore, Billerica, MA, USA). Un-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA) quantified the signal intensities. Data were normalized to appropriate references (GAPDH for cytoplasmic protein and Histone 3 for nuclear protein).
10
Protein Extraction and Western Blot Analysis
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Whole cell protein extraction, cytosolic protein extraction, and nuclear protein extraction were extracted according to previous studies (Pan et al., 2014 (link); Zhang et al., 2016a (link)). Equal protein amounts were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% skim milk for 2 h at room temperature and then incubated with primary antibodies against caspase-3 (1:400, cat #9661, Cell Signaling), SIRT1 (1:200, cat #SC-15404, Santa Cruz), HMGB1 (1:1000, cat #3935S, Cell Signaling), TLR4 (1:200, cat #sc-30002, Santa Cruz), Myd88 (1:200, cat #sc-11356, Santa Cruz), Nf-κB p65 (1:200, cat #sc-372, Santa Cruz), β-actin (1:3000, cat #AP0060, Bioworld Technology, Minneapolis, MN, USA), and Histone H3 (1:3000, cat #BS7416, Bioworld Technology) overnight at 4°C. The membrane was incubated with horseradish peroxidase (HRP)-conjugated IgG for 2 h at room temperature. Detection was performed by enhanced chemiluminescence solution (Thermo Fisher Scientific, Waltham, MA, USA). Band density was quantified with UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).
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