Total protein was extracted from tissues or cells using the Protein Extraction kit (Beyotime Biotechnology, Shanghai, China). Samples were separated by 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were incubated in diluted primary antibodies overnight at 4 °C (the primary antibodies are listed in Supplementary Table S3 ). After incubation with secondary antibodies (Goat anti-rabbit or Goat anti-mouse, 1:5000 respectively; Proteintech, Hangzhou, China), membranes were visualized with Quantity One imaging software (Bio-Rad, California, USA). ImageJ software v.1.48 was used to measure the relative integrated density values (IDVs) based on Tubulin (1:4000; Proteintech, Hangzhou, China) as an internal control.
Quantity one imaging software
Manufactured by Bio-Rad
Sourced in United States
Quantity One is an imaging software developed by Bio-Rad. It is designed to analyze and quantify data from various imaging techniques, including gel electrophoresis, Western blotting, and other applications. The software provides tools for image acquisition, processing, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescent detection system, by Santa Cruz Biotechnology (7 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Chemicdoc xrs system, by Bio-Rad (5 mentions)
Peroxidase conjugated anti rabbit igg, by Santa Cruz Biotechnology (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Chemidoc xrs system, by Bio-Rad (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Chemi doc xrs imaging device, by Bio-Rad (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Ecl plus western blotting detection reagent, by Cytiva (3 mentions)
Zeta probe gt nylon membrane, by Bio-Rad (3 mentions)
Protein extraction kit, by Beyotime (2 mentions)
Ripa protein extraction reagent, by Beyotime (2 mentions)
γ tubulin, by Bio-Rad (2 mentions)
Anti p66shc, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
73 protocols using quantity one imaging software
1
Western Blot Analysis of Protein Expression
2
Quantitative Western Blot Analysis of HER-2
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Western blot analysis was carried out on 10% SDS–PAGE. Briefly, HER-2 proteins were electrotransferred onto nitrocellulose filter. After blocking for 1 h in PBS with 0.1% Tween 20 (PBST) and 5% skim milk, the membranes were incubated overnight with specified primary antibody in PBST containing 5% BSA. The primary antibodies used included HER-2 (Cell Signaling Technology) and GAPDH (Bioworld Technology). Assay was carried out by the use of second antibody and developing agent. Western blot was scanned and band intensity was quantified by using Quantity One Imaging Software from Bio-Rad.
3
Global Imaging and Tissue Sampling for Aqueous Outflow
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Global imaging was performed, as described previously (Cha et al., 2016 (link)) to visualize the outflow pattern along the entire TM and episcleral veins. Each fixed globe (n = 2) was cut into anterior and posterior segments through the equator, followed by careful removal of the lens, vitreous body, iris, cornea (with a 10-mm trephine), and excess conjunctiva. Anterior segments were imaged en face on both the TM and scleral surfaces with a fixed exposure time (5 s) using a 300 mm lens on a 4000 MP VersaDock imaging system (Bio-Rad Laboratories, Hercules, CA, United States) with Quantity One imaging software (Bio-Rad Laboratories).
From global images, flow-type areas were designated qualitatively based on the relative amount of fluorescence as follows: 1) high-flow, bright fluorescence on both anterior (episcleral veins) and posterior (TM) sides of anterior segments; 2) low-flow, intermediate fluorescence on both posterior and anterior sides; 3) non-flow, no visible fluorescence on either anterior or posterior sides. Global images of these eyes have been published previously (Swain et al., 2021 (link)). Two radial wedges (2 × 2 × 2 mm) of tissue from each flow-type area were shipped to Cleveland Clinic (Cleveland, OH, United States) for SBF-SEM.
From global images, flow-type areas were designated qualitatively based on the relative amount of fluorescence as follows: 1) high-flow, bright fluorescence on both anterior (episcleral veins) and posterior (TM) sides of anterior segments; 2) low-flow, intermediate fluorescence on both posterior and anterior sides; 3) non-flow, no visible fluorescence on either anterior or posterior sides. Global images of these eyes have been published previously (Swain et al., 2021 (link)). Two radial wedges (2 × 2 × 2 mm) of tissue from each flow-type area were shipped to Cleveland Clinic (Cleveland, OH, United States) for SBF-SEM.
4
Quantitative Western Blotting of PXR and CYP3A
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Western blotting experiments were performed using SDS-PAGE electrophoresis for total protein fractions of mouse liver samples as described elsewhere32 (link). Primary antibodies used for detection were anti-PXR (PA5-41170) and anti-CYP3A11 (PA1-343, Thermo Fisher Scientific). Protein band intensity on PVDF membranes was quantified using Quantity One imaging software (Bio-Rad, Hercules, CA, USA). Protein expression was normalized to expression of β-actin (A5316, Sigma–Aldrich). The results are presented as relative change in protein expression compared to corresponding controls in the same interval after PCN application. The data are shown as the mean ± SD acquired from five biological samples per group. For Western blotting experiments with primary human hepatocytes and cancer cell lines lysates, β-actin (A5316, Sigma–Aldrich), PXR (PA5-41170, Thermo Fisher Scientific), CYP3A4 (PA1-343, Thermo Fisher Scientific), and RXRα (5388, Cell Signaling Technology, Danvers, MA, USA) primary antibodies were used.
5
Protein Extraction and Western Blot Analysis of BMSCs
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BMSCs were lysed using RIPA protein extraction reagent (Beyotime, Beijing, China) supplemented with protease inhibitors and phosphatase inhibitors (Beyotime). The concentrations of extracted proteins were measured by a BCA Protein Assay Kit (Beyotime). The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After blocking with 5% bovine serum albumin (BSA) for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies are listed in Table S2 . Subsequently, the membranes were incubated at 37 °C with horseradish peroxidase (HRP)-conjugated goat anti-mouse or HRP-conjugated goat anti-rabbit for 2 h. Protein bands were visualized with an Enhanced Chemiluminescence Kit (Beyotime) and analyzed using Quantity One Imaging Software (Bio-Rad, CA, USA).
6
Quantification of NF-κB Expression
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The expression of NF-κB was analyzed by Western blotting. We separated 50 μg protein on 6.25% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) using the mini-PROTEAN 3 electrophoresis cell (Bio-Rad Laboratories, Inc., Hercules, California, USA) according to the manufacturer’s protocol. After being transferred to a nitrocellulose membrane, the proteins were blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST). The membrane was then incubated with anti- NF-κB antibody for 1 h, washed four times with TBST, and incubated for 1 h with a peroxidase-conjugated secondary antibody (1:1000). After washing the membrane four times with TBST buffer, the blot was developed using ECL chemiluminescence reagents (Millipore immobilonTM HRP Substrate) and imaged. The immunoreactive bands on the autoradiography films were scanned with a calibrated densitometer ChemiDocTM XRS+ (Bio–Rad imaging System) and quantified using QuantityOne imaging software (Bio–Rad Laboratories, Hercules, CA). Equal loading of proteins onto the gel was confirmed by the immunodetection of GAPDH.
7
Rg1 Modulates Cell Apoptosis Pathways
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To assess the effects of Rg1 on expression of cell apoptosis-associated molecules, quantitative RT-PCR assay was conducted. Total cellular RNAs were extracted using commercial TRIzol regents (Takara, Dalian, China). The Reverse Transcription kit (Takara) was then used to generate complementary DNAs (cDNAs) using on the protocols of manufacturer. The synthesized cDNAs were amplified as the templates using the Sybr green I (Takara) and ABI Fast real-time PCR system (ABI, Foster City, CA, USA). Then, the PCR assay was conducted with 35 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 30 s. Table 1 shows the specific primers amplifying targeting genes, including cleaved caspase 3, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), SIRT3, SOD2, and β-actin. Images of amplified products were analyzed with Quantity One Imaging Software (Bio-Rad, CA, USA) and with the comparative threshold cycle (2−ΔΔCT) method [21 (link)]. The RT-PCR assay was conducted at least 6 times in each group.
8
Quantitative Assessment of RT-PCR Amplicons
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RT-PCR samples were electrophoretic on 1% agarose gel and photographed under ultraviolet light using BioRad imager. BioRad quantity one imaging software was used to quantify the strip strength.
9
NF-κB Expression Analysis in BV-2 Cells
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To analyze the expression of NF-κB, BV-2 cells were lysed in a buffer containing 20 mM Tris–HCl (pH = 7.4), 1 mM EDTA, 140 mM NaCl, 1% (w/v) Nonidet P-40, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 10 μg/mL aprotinin. The cell lysates were separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were soaked in blocking buffer (1× Tris-buffered saline (TBS), 0.1% Tween 20, and 5% non-fat dry milk) for 1 h and incubated overnight at 4 °C with primary antibodies against NF-κB (Abcam, Cambridge, UK; 1:1000) and β-actin (Cell Signaling Technology, Danvers, MA, USA; 1:1000). The blots were developed using peroxidase-conjugated anti-mouse and anti-rabbit IgG and a chemiluminescent detection system (Santa Cruz Biotechnology, Dallas, Texas, USA). The bands were imaged using a ChemicDoc XRS system (Bio-Rad,) and analyzed using Quantity One imaging software (Bio-Rad). Each sample was assayed in triplicate and each experiment was performed twice.
10
Protein Extraction and Immunoblotting Analysis
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Protein extracts were prepared by washing the cells in PBS and incubating for 20 min in ice-cold lysis buffer supplemented with protease inhibitor cocktail, as previously reported 16 (link). After sonication for three times for 10 s, proteins were quantified by Bradford method and equal amount of proteins (40 μg) was separated by SDS-PAGE and then electrotransferred to a nitrocellulose membrane for the detection with specific antibodies. The blots were developed using the alkaline phosphatase colorimetric or ECL chemiluminescent labeling systems. Optical densities of the bands were analyzed with Quantity One Imaging Software from Bio-Rad Laboratories. The correct protein loading was verified by means of both Ponceau red staining and immunoblotting for actin. The results showed in the figures are representative of almost four independent experiments with similar results.
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