Chondrocytes treated with Wogonin (50 μM) under stimulation with IL-1β (10 ng/ml, 48 h) were harvested, washed with cold PBS and lysed in ice-cold RIPA buffer and lysate were prepared as described previously [5 (link)]. Equivalent amounts of lysate protein (35 μg) were resolved by 10% SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, USA) and the blots were incubated with primary antibodies diluted in 2% blocking buffer for overnight at 4°C. Blots were then incubated with horse radish peroxidase-conjugated secondary antibody, followed by washing with TBST. Blot were developed using Luminata Western HRP substrate (EMS Millipore) and the antibody reactive proteins were visualized by chemiluminescence and imaged using the Pxigel imaging system (Syngene, Frederick, MD).
Pxi gel imaging system
Manufactured by Syngene
Sourced in United Kingdom, United States
The PXi gel imaging system is a versatile instrument designed for capturing high-quality images of nucleic acid and protein gel electrophoresis samples. It utilizes advanced imaging technology to provide clear and accurate results for various applications in life science research.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (3 mentions)
Anti ha antibody, by Thermo Fisher Scientific (2 mentions)
Protease and phosphate inhibitors, by Thermo Fisher Scientific (2 mentions)
Anti bmp2 monoclonal detection antibody conjugated to horseradish peroxidase, by Sino Biological (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Mini protean, by Bio-Rad (1 mentions)
Xp rabbit mab, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Prism 5, by GraphPad (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Pierce bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Luminata western hrp substrate, by Merck Group (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using pxi gel imaging system
1
Wogonin Modulates Chondrocyte Response
2
Protein Quantification and Western Blotting
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Cells were washed with ice cold PBS and lysates prepared as described17 (link). Total protein was quantified using a BCA assay (ThermoFisher) according to the manufacturer’s instructions. 50 μg per lane were loaded for ORAI1 gels, while 30 μg per lane were loaded for all others. 4–20% Mini-Protean TGX Pre-cast gels (BioRad) and iBlot PVDF kits (ThermoFisher) were used for SDS-PAGE and transfer respectively. All antibodies were diluted in 3% milk/1%Tween/TBS, primary antibodies were incubated overnight and secondary antibodies incubated for 1 h. Membranes were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and a PXi gel imaging system (Syngene, UK).
3
Insulin Bioactivity Assay in Hepatocytes
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The bioactivity of the stored insulin samples has been assessed using two hepatocytes cellular models, in which insulin efficiency on insulin receptor (IR) and Akt phosphorylation was detected and quantified as previously reported [34 (link)]. Briefly, starved cells were stimulated with 10-8M insulin for 15 min at 37°C and then immediately frozen by immersion in liquid nitrogen to stop insulin signaling. Cells were then lysed in ice-cold RIPA buffer and equal amounts of proteins were resolved by 5–20% gradient SDS-PAGE followed by Western blotting onto nitrocellulose membranes for immunodetection of insulin receptor and Akt phosphorylation. Constitutive expression and phosphorylation of proteins of interest were detected with specific primary antibodies and HRP-conjugated secondary antibodies using chemoluminescence. Quantifications were performed using the PXi gel imaging system and the Genesys/Genetools softwares (Syngene, Cambridge, UK).
4
Western Blot Analysis of BMP2 Signaling
5
Western Blot Analysis of OA Chondrocytes
6
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cells or ex vivo liver tissues (30 µg) using ice-cold RIPA buffer (150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% deoxycholicacide, 50 mM Tris-HCl pH 7.4, 0.5 M EDTA, 1 mM vanadate, 10 mM Naf, 1 mM PMSF and 1 tablet of cOmplete™ Protease Inhibitor Cocktail (Roche)). The supernatant was collected after centrifugation (10 min at 12,000× g) and the protein content was measured using a BCA protein assay with bovine serum albumin as standard (Pierce Biotechnology). Then, 10 µg of proteins was submitted to SDS-PAGE (5–20% gradient gels) and transferred on nitrocellulose membranes (Amersham, Dübendorf, Switzerland), which were then blocked in polyvinyl alcohol for 1 min. Next, proteins of interest were probed with specific primary antibodies overnight at 4 °C and, after several washings with TBS 0.1% Tween 20, with proper horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The signal was detected by PXi gel imaging system (Syngene, Cambridge, UK) and quantified with ImageJ software.
7
Western Blot Analysis of Protein Expression
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Primary cells were grown to confluency and lysed in RIPA buffer containing Halt protease and phosphatase inhibitors (1:1000). Protein concentration was determined using the PierceTM bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific). In total, 20 µg protein was separated on 8% Laemmli SDS-PAGE, and transferred onto polyvinylidene difluoride membrane (Immun-Blot® PVDF membrane, Bio-Rad). Membranes were blocked in a 5% w/v milk (Sigma) and 2% w/v BSA in TBST solution before incubation with primary antibody overnight at 4 °C. Primary antibodies used are listed in Table S1 . Secondary HRP-conjugated antibody (Bio-Rad) (1:5000) was added and immunoreactivity detected using the ECL or ECL plus Western Blotting substrate (PierceTM). Membranes were developed by exposure to Amersham Hyperfilm ECL (GE healthcare) or using the PXi gel imaging system (Syngene). Image J was used for quantification of all Western blot bands, using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. Statistical analysis was performed on the raw densitometry values using Graphpad Prism 5, p < 0.05. A Student’s Paired T test or a Student’s Unpaired T test were used to assess two samples. A one-way ANOVA/Repeated Measures ANOVA was used for more than two samples with Tukey’s Multiple Comparison Test (p < 0.05).
8
Western Blotting for Protein Analysis
9
Validation of PD-L1 scFv Expression
10
Western Blot Analysis of Chondrocyte Proteins
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After treatment with DMSO or IL-1β 1 ng/mL for 24 h, or pretreatment with ICA (10−9 M) for 2 h followed by IL-1β (1 ng/mL) for 24 h, chondrocytes were harvested and proteins isolated using an extraction kit (Beyotime Institute of Biotechnology, Jiangsu, China), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membranes (Bio-Rad, USA). Membranes were blocked in 5% bull serum albumin (Sigma, TX, USA) for 2 h and then incubated with primary antibodies overnight at 4 °C. Blots were then incubated with horseradish peroxidase-conjugated secondary antibody, followed by washing with TBST (Bio-Rad, USA). Blots were developed using Luminata Western HRP substrate (EMS Millipore, USA), and antibody reactive proteins were visualized by chemiluminescence and imaged using the Pxigel imaging system (Syngene, Frederick, MD).
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