Total protein was isolated from the wound tissue obtained at indicated times with RIPA (Aspen Inc.) following the manufacturer’s instruction. The protein concentrations were determined using the BCA (Aspen Inc.). Equal amounts of protein from each tissue were separated by SDS-PAGE (Aspen Inc.), transferred to a PVDF membrane, and incubated with 5% milk for 1 h at room temperature. The membranes were incubated overnight at 4 °C with rabbit anti-phosphorylated-ERK (1:1,000; Cell Signaling Technology), rabbit anti-ERK (1:2,000; Cell Signaling Technology), rabbit anti-phosphorylated-AKT (1:1,000; Cell Signaling Technology), rabbit anti-AKT (2:1,000; Cell Signaling Technology), rabbit anti-SPRED1 (1:1,000; ABCAm), mouse anti-VEGF (1:500; ABCAm), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (1:1,000; ABCAm). Next, anti-rabbit IgG (1:10,000; Aspen, Inc.) and anti-mouse IgG (1:10,000; Aspen, Inc.) were used as second antibodies at 1:2,000 for 30 min. Quantification of the protein bands were performed with AlphaEaseFC (Alpha Innotech, San Leandro, CA) software. Sample analyses were performed on day 9.
Alphaeasefc
Manufactured by Bio-Techne
Sourced in United States
AlphaEaseFC is a software application for image analysis and quantification. It provides tools for capturing, processing, and analyzing digital images from various sources, including Western blots, gels, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Radioimmunoprecipitation assay (ripa), by Aspen (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Enhanced chemi luminescence (ecl), by Wuhan Servicebio Technology (2 mentions)
Bca kit, by Thermo Fisher Scientific (2 mentions)
As1012, by Aspen (2 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Mouse anti vegf, by Abcam (1 mentions)
Bicinchoninic acid (bca), by Aspen (1 mentions)
Rabbit anti phosphorylated erk, by Cell Signaling Technology (1 mentions)
Rabbit anti erk, by Cell Signaling Technology (1 mentions)
Rabbit anti phosphorylated akt, by Cell Signaling Technology (1 mentions)
Rabbit anti glyceraldehyde 3 phosphate dehydrogenase, by Abcam (1 mentions)
Sds page, by Aspen (1 mentions)
Hrp conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Nuclear extraction kit, by Active Motif (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Alphaease fc imaging system, by Bio-Techne (1 mentions)
52 protocols using alphaeasefc
1
Protein Analysis of Wound Tissue
2
Western Blot Analysis of PrPSc
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Following PK digestion, the samples were mixed with an equal volume of a 2-butanol:methanol mixture (5:1), and PrPSc was precipitated by 20,000 ×g centrifugation. The pellets were resuspended in Laemmli sample buffer and boiled for 5 min. Samples were electrophoresed on NuPAGE Novex 12% Bis-Tris gels and NuPAGE MOPS-SDS running buffer in accordance with the manufacturer's instructions (Life Techonologies, Carlsbad, CA, USA). The proteins were transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). The blotted membrane was incubated with anti-PrP monoclonal antibodies T2, 6H4 (Prionics, Zurich, Switzerland), and SAF84 (Bertin Pharma, Montigny le Bretonneux, France) at 4°C overnight. After washing with PBS containing Tween 20 twice, the membrane was incubated with HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 60 min at R/T. Signals were developed with a chemiluminescent substrate (SuperSignal; Thermo Fisher Scientific). For semi-quantitation, blots were imaged using a Fluorchem system (Alpha Innotech, San Leandro, CA, USA) and analyzed using image reader software (AlphaEaseFC; Alpha Innotech) according to the manufacturer's instructions.
3
Western Blot Analysis of Apoptosis and Autophagy Markers
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Neuronal cells were seeded at 30,000 cells/cm2, differentiated and treated in collagen-coated 6-well plates. Total proteins were extracted (Nuclear Extraction Kit, Active Motif, Carlsbad, CA, USA) and concentrations were determined by bicinchoninic acid quantification (BCA protein assay kit, Pierce Biotechnology Inc., Rockford, IL, USA). Equal amounts of protein were loaded onto a 12% SDS-polyacrylamide gel. After electrophoretic separation (125 V, for 1.5 h), proteins were transferred onto PVDF membranes (0.22 μm pore size, BioRad) at 25 V overnight. The membranes were blocked for 30 min to 1 h and incubated overnight at 4 °C with primary antibodies anti-Bax, anti-Bcl-2, anti-LC3, anti-p62 (Progen Biotechnik GmbH, GP62-C) and anti-actin, (1:200, 1:100, 1:500, 1:1000, and 1:2000, respectively). The blots were then incubated with the appropriate peroxidase-conjugated secondary antibody (1:10,000) for 2 h at room temperature and finally developed with an enhanced chemiluminescence substrate solution (ThermoFisher Scientifics, Ottawa, ON, Canada). Immunopositive chemiluminescent signals were visualized with the AlphaEase FC imaging system (Alpha Innotech, San Leandro, CA, USA) and analyzed using AlphaEase FC (Alpha Innotech San Leandro, CA, USA) and ImageJ (https://imagej.nih.gov/ij/ ) software packages.
4
Western Blot Analysis of Tissue Proteins
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Total tissue proteins were extracted for Western blot analysis as described in a previous study.27 (link) Briefly, approximately 20 μg of protein from each sample was separated on 10% SDS-PAGE gels and then transferred to PVDF membranes. The membranes were incubated with primary antibodies against PPARγ, TGF-β1, α-SMA, Collagen I and β-actin overnight at 4°C. Afterwards, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The protein bands were analyzed with AlphaEaseFC (Alpha Innotech, San Leandro, CA, USA).
5
Immunoblotting Analysis of CaMKII and GST
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For immunoblot analysis, protein was transfered onto PVDF membrane (Goodell et al., 2017 (link)) and detected using antibodies specific for CaMKIIα (CBα2) or GST. Briefly, 10 μg of total protein was subjected to SDS-PAGE on 7.5% polyacrylamide gels made in house and transferred to PVDF membrane for 1–2 hrs at 4°C. In GluN2B binding assays, GST was used to assess total protein and bound CaMKII levels were determined by the ratio of bound to total CaMKII. Blots were developed using chemiluminescence (Super Signal West Femto,Thermo-Fisher) and imaged using the Chemi-Imager 4400 system (Alpha-Innotech). Densitometry was calculated in AlphaEaseFC (Alpha-Innotech) (Barcomb et al., 2014 (link); Goodell et al., 2014 (link), 2016 (link)). The relative immuno-detection value (IDV) was normalized as a percent of the average of WT conditions for the same blot, which was set at a value of one to allow comparison between multiple experiments. Sample size is indicated by individual data points on graph and statistical analysis is detailed in figure legends.
6
Western Blot Analysis of Prion Protein
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Samples were electrophoresed on NuPAGE Novex 12% Bis-Tris gels with NuPAGE MOPS-SDS running buffer in accordance with the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA). Proteins were transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). The blotted membrane was incubated with anti-PrP mAb T2-HRP [14 (link)] at 4°C overnight. After washing twice with PBS containing 0.05% (v/v) Tween 20 (PBS-T), signals were developed with a chemiluminescent substrate (SuperSignal; Thermo Fisher Scientific K.K., Yokohama, Japan). For semi-quantitation, blots were imaged using a Fluorchem system (Alpha Innotech, San Leandro, CA, USA) and analyzed using image reader software (AlphaEaseFC; Alpha Innotech) according to the manufacturer’s instructions.
7
Western Blot Analysis of Signaling Proteins
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Cell extracts were prepared using ice-cold RIPA lysis buffer (EMD Millipore, Billerica, MA, USA) containing a protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The protein extracts were denatured in a boiling water bath for 5 min and then resolved by SDS-PAGE. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore), which were probed with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at 1:4,000 dilutions (Cell-Signaling Technology, Danvers, MA, USA) for 1 hr at room temperature. The signal of the targeted protein was developed using a chemiluminescence kit (EMD Millipore). Primary antibodies used include those against PTEN (catalog no. 9188), p-AKT (catalog no. 4060), AKT (catalog no. 4691), p-STAT3 (catalog no. 9131), STAT3 (catalog no. 4904), p-STAT6 (catalog no. 9361), STAT6 (catalog no. 9362) (Cell-Signaling Technology, Danvers, MA, USA), and GAPDH (EMD Millipore, catalog no. MAB374). To quantify the immunoblot images on unsaturated bands, densitometric analysis was performed using AlphaEaseFC (Alpha Innotech) followed by statistical evaluation.
8
Quantifying Protein Expression Levels
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The GLUT1, FASN, and matrix metalloproteinase-2 (MMP2) expression levels were measured using Western blot analysis. Samples were lysed with a lysis buffer containing a protease inhibitor cocktail (G2006, Servicebio), and the protein concentrations were determined using a protein assay kit (G2006, Servicebio). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with 20 μg of protein in each sample using 4% to 15% Mini Protean TGX precast gels (Servicebio). The following primary antibodies were used: anti-GLUT1 (AB652, 1:1000, Abcam), anti-FASN (AB22759, 1:1000, Abcam), and anti-MMP2 (GB11130, 1:1000, Servicebio). The bands were detected using a Western blotting detection system (Epson, Japan) and band intensity was calculated via densitometry using AlphaEaseFC (Alpha Innotech, USA).
9
Quantifying NLRP3 and P2X7 Proteins
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First, the hippocampus was lysed using a lysis buffer (RIPA buffer with proteinase inhibitors). Then, samples were quantified by the BCA protein quantitative detection kit before Western blotting. Equal amounts of total protein extracts were separated by SDS-PAGE and electro-transferred to PVDF membranes (0.45 μm). Then, the anti-NLRP3 and anti-P2X7 was used as the primary antibody, followed by incubation with appropriate HRP-conjugated secondary antibodies (GB23303, Servicebio). Proteins were detected with ECL (G2014, Servicebio) and quantified by densitometry using analytic software (alphaEaseFC, Alpha Innotech). Results were normalized using anti-β-actin antibody densitometric values.
10
Wound Healing Assay in Cells
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Cells were cultured in RPMI 1640 containing 1/10 FBS. After cultivating for 24 h, a sterile plastic 200 mL micropipette tip was used to scratch the monolayer. Then, the monolayer was washed, and incubated in serum-free RPMI 1640. At 0 and 24 h, a low-magnification fluorescence microscope (Olympus IX71, Tokyo, Japan) was applied to photograph the cells. AlphaEase FC (Version 4.0, Alpha Innotech, San Francisco, CA, USA) was adopted to measure the widths of the wound lines.
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