After cellular proteins isolation using a Protease Inhibitor Cocktail and Protein Phosphatase Inhibitor (Beijing Solarbio Science & Technology Co., Ltd.)-supplemented Radio-Immunoprecipitation Assay (RIPA) buffer (Gibco; Thermo Fisher Scientific), the protein samples were loaded onto a sodium dodecyl sulfate polyacrylamide gel and subsequently blotted onto a PVDF membrane. Primary antibodies (Abs) were applied and incubated overnight in 5% bovine serum albumin at 4 °C. Western blotting analysis was conducted using ImageJ software (Bio-Rad Laboratories, Inc.). The primary Abs used were β-actin Ab (1:2000, 3700S, Cell Signaling Technology, Inc.), ATM Ab (1:2000, ab201022, Abcam), p-ATM Ab (1:1000, ab81292, Abcam), cleaved caspase-3 Ab (1:1500, ab32042, Abcam), cleaved caspase-9 Ab (1:1500, ab2324, Abcam), 53BP1 Ab (1:1000, ab175933, Abcam), H2AX Ab (1:1000, 7631S, Cell Signaling Technology), and p-H2AX Ab (1:500, 9718S, Cell Signaling Technology). For secondary Abs, the following were used: HRP-linked anti-mouse IgG (1:8000, 7076P2) and HRP-linked anti-rabbit IgG (1:5000, 7074P2), both from Cell Signaling Technology.
Imagej software
Manufactured by Bio-Rad
Sourced in United States, China
ImageJ is a free, open-source image processing software developed by the National Institutes of Health. It is designed for analyzing and processing digital images, including those acquired from scientific instruments.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (20 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Ab205718, by Abcam (6 mentions)
Polyvinylidene fluoride membrane, by Merck Group (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Gapdh, by Abcam (5 mentions)
Image lab software, by Bio-Rad (5 mentions)
Ab8245, by Abcam (5 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Ab8227, by Abcam (4 mentions)
Ab6721, by Abcam (4 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (4 mentions)
Ab32042, by Abcam (3 mentions)
Ecl reagent, by Thermo Fisher Scientific (3 mentions)
β actin, by Abcam (3 mentions)
E cadherin, by Cell Signaling Technology (3 mentions)
Protease inhibitor, by Thermo Fisher Scientific (3 mentions)
Chemiluminescence imaging system, by Bio-Rad (3 mentions)
Gapdh, by Proteintech (3 mentions)
139 protocols using imagej software
1
Protein Isolation and Western Blotting
2
Western Blot Analysis of Apoptosis and EMT Markers
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The GC cells was separated using RIPA lysate (89901, Thermo Fisher Scientific, Inc.), and the protein concentration was determined using a Protein Assay kit (A53227, Thermo Fisher Scientific, Inc.). A volume of 30 µg proteins of each tissue was separated on 10% SDS-polyacrylamide gels and transferred to a PVDF membrane (HVLP04700, EMD Millipore), which the membrane was then washed with TBS and blocked with 5% non-fat milk at 37°C for 1 h and shaken for 2 h at room temperature. The membrane was then incubated with the following primary antibodies: Anti-Bcl2 (1:1,000; 26 kDa; rabbit; ab32124), anti-procaspase-3 (1:10,000; 35 kDa; rabbit; ab32499), anti-cleaved caspase-3 (1:500; 17 kDa; rabbit; ab32042), Vimentin (1:1,000; 54 kDa; rabbit; ab92547), N-cadherin (1 µg/ml; 130 kDa; rabbit; ab18203), E-cadherin (1:10,000; 97 kDa; rabbit; ab40772) or GAPDH (1:10,000; 36 kDa; rabbit; ab181602) (all from Abcam) overnight at 4°C. The target band was incubated with a goat anti-rabbit IgG H&L (HRP) (1:5,000; ab205718; Abcam) for 2 h at room temperature. Finally, the signals were measured by ECL reagent (6883, Cell Signaling Technology, Inc.), and the gray values of the bands were analyzed and counted using ImageJ software (version 5.0, Bio-Rad Laboratories, Inc.) (16 (link)).
3
Angiogenesis Proteome Profiling in Cardiac Regeneration
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Angiogenesis proteome profiling was carried out with 100 µg protein per sample (before cell injection, and 12 and 24 h as well as one month later) on Proteome Profiler Human Angiogenesis Array Kit (R&D Systems ARY007). Protein lysates were isolated as described above. Myocardial protein isolates were handled as described in the protocol guide. Chemiluminescent signal pixel densities were detected on X-ray films (Bio-Rad Molecular Imaging System) and analysed by ImageJ software (developed by the Laboratory of Optical and Computational Instrumentation, University of Wisconsin, NIH, U.S.). Myocardial tissue proteome was further analysed in the String functional protein association network database for Sus scrofa species (Szklarczyk et al., 2015 (link)). To assess relevance in human cardiac regeneration, the same protein library was analysed in Ingenuity Pathway Analyses.
4
Protein Expression Analysis of ESCC Cells
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ESCC cells were lysed at 4°C in radio immunoprecipitation assay buffer (Solarbio) mixed with protease and phosphatase inhibitors. The concentration of total protein was assessed using Ultra-micro ultraviolet spectrophotometer (Thermo, USA). Proteinlysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immun-Blot PVDF membranes (Solarbio). and immersed in a blocking solution containing 5% non-fat milk and 0.1% Tween-20 for 1 h.
After blocking, membranes were incubated with speci c primary antibodies against Bcl-2 (Beyotime biotechonology), Bax (Abcam), MMP-9 (Abcam), MMP-2( Santa Cruz) and β-actin (Abcam) at 4°C overnight, and then with secondary antibodies for 2 h at room temperature. After washing, The blots were developed with ECL reagents (Thermo), quanti ed by densitometry, then analyzed using the Image J software (BIO-RAD Company, USA), and normalized to the corresponding β-actin bands.
After blocking, membranes were incubated with speci c primary antibodies against Bcl-2 (Beyotime biotechonology), Bax (Abcam), MMP-9 (Abcam), MMP-2( Santa Cruz) and β-actin (Abcam) at 4°C overnight, and then with secondary antibodies for 2 h at room temperature. After washing, The blots were developed with ECL reagents (Thermo), quanti ed by densitometry, then analyzed using the Image J software (BIO-RAD Company, USA), and normalized to the corresponding β-actin bands.
5
Quantitative Analysis of Slug and P4HA3 Proteins
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Cells were harvested and lysed in an Radioimmunoprecipitation assay (RIPA) buffer. Protein lysates of 30 µg were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by western blot analysis. The antibodies used were as follows: rabbit monoclonal anti-Slug (1:200, sc-166476, Santa Cruz, Cambridge, UK), anti-P4HA3 (1:1000, ab101657, Abcam), and anti-β-actin (1:1000, ab3280, Abcam, Cambridge, UK). Antigen–antibody complexes were detected by electrochemiluminiscence Western blotting detection reagent (GE Healthcare, Marlborough, Massachusetts). Band densitometry was performed using ImageJ software (version 1.48, Bio-Rad, Hercules, California, USA).
6
Quantitative Analysis of MMP-13 Protein
7
Western Blotting Procedure for Protein Analysis
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Western blotting for this study was similar to the procedure in our previous study 60 (link). Briefly, the cultured cells with the indicated treatment were harvested and lysed in cell lysis buffer (Beyotime, #P0013B) containing a protease and phosphatase inhibitor cocktail (Beyotime, #P1045). The lysates were normalized to equal amounts of protein using the bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, #P0011). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 8%-12% acrylamide) then transferred to polyvinylidene difluoride membranes (Millipore, #ISEQ00010) followed by blocking with 5% skimmed milk. The membranes were incubated with primary antibodies overnight, followed by incubation with anti-mouse (1:6000 dilution), anti-rabbit (1:8000 dilution), or anti-goat (1:5000 dilution) horseradish peroxidase (HRP)-conjugated secondary antibodies. The blots were visualized with enhanced chemiluminescence (ThermoFisher Scientific, #34580) and imaged with a Tanon 5200 image analysis system (Tanon, Shanghai, China). The protein bands were quantified with the ImageJ software (Bio-Rad).
8
Western Blot Analysis of EMT Markers
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RIPA lysis buffer including protease inhibitor (Madison, WI, USA) was utilized to isolate total protein. The proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, NY, USA). Subsequently, the membrane was blocked with 5% skim milk for 1 h. Then the membrane was incubated with primary antibodies overnight at 4 °C, and hatched with secondary antibodies for 1 h at room temperature. With an ECL Kit (Millipore, NY, USA), the proteins were visualized and tested utilizing an ImageJ software (Bio-Rad, Hercules, CA). Western blotting analysis was executed utilizing the primary antibodies: anti-E2F7 (1: 1000, ab245655, Abcam, MA, USA), E-cadherin (1:1000, #14472, Cell Signaling Technology, Inc.), N-cadherin (1:1000, #13116, CST), Vimentin (1:1000, #5741, CST), Snail (1:1000, #3879, CST), GAPDH (1:5000, #5174, CST) and the secondary antibodies (1:5000, #7076 or #7074, CST). The expression of GAPDH was a control.
9
Transwell Invasion and Migration Assay
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The invasion and migration of OSCC cells were evaluated by applying Transwell inserts pre-coated with or without Matrigel. Briefly, the transfected cells (1× 105) in 100 μl of serum-free medium were seeded on the top chamber of 24-well plates; whilst 600 μl RPMI-1640 medium (supplying with 10% FBS) was added to the lower chamber. Incubating for 24 h, the cells were fixed with 4% polyoxymethylene for 30 min and dyed with 0.1% crystal violet for 10 min at 37 °C. Finally, a light microscope at magnification × 200 was utilized to visualize the outcomes. The number of cells was analyzed on ImageJ software (Bio-Rad, Hercules, CA). Each assay was executed in triplicate.
10
Western Blot Analysis of PTER in Hepatoma Cells
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Western blotting was carried out as described previously (7 (link)). Briefly, SMMC-7721 hepatoma cells were treated with various concentrations (5, 25, 50, or 100 µM) of PTER for 24 h. PTER (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulphoxide (DMSO); DMSO alone was used to treat cells in the control group. After washing with PBS, cells were then lysed using RIPA buffer, which was mixed with the Halt Phosphatase and Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc., Waltham, MA, USA). After the protein concentration was quantified, 70 µg of protein was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane using an Electrophoresis Transfer System (Bio-Rad). Subsequently, membranes placed on a swing table were blocked with TBS-Tween and 5% dry milk and were then probed with anti-MTA1, p53 or Ac-p53 (Abcam, Cambridge, UK) primary antibodies overnight at 4°C. Respective secondary antibodies (Abcam) were used at 1:1,000–1:2,000 (v/v) dilutions in TBS-Tween and 5% fat-free powdered milk for 1 h at room temperature; β-actin antibodies were used as a loading control. Finally, blots were visualized by SuperSignal West Pico enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the density of the blots was quantified using Image J Software (Bio-Rad).
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