Total protein was extracted from macrophages using pre-cold radio immune precipitation assay buffer (Boster, Wuhan, China) in the presence of 1% protease and phosphatase inhibitors (Boster, Wuhan, China). The cell lysates were centrifuged at 12,000 g at 4°C for 10 min, mixed with a loading buffer, and boiled for 5 min at 98°C. The obtained supernatants were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were subsequently blotted onto the poly (vinylidene fluoride) membrane (Millipore, Bedford, USA). The membranes were then blocked with 5% bovine serum albumin in TBST buffer and incubated with the following primary antibodies overnight at 4°C. After the solutions were washed three times with TBST buffer, the membranes were incubated with (HRP)-conjugated secondary antibodies (1:5000, Beyotime) for 1 h at room temperature and then washed with TBST for three times. The blotting signals were detected using an enhanced chemiluminescence reagent (Beyotime, Shanghai, China), and quantified using ImageJ.
Protease and phosphatase inhibitors
Manufactured by Boster Bio
Sourced in China
Protease and phosphatase inhibitors are chemical compounds used in laboratory settings to prevent the degradation of proteins and disruption of cellular signaling pathways. These inhibitors work by blocking the activity of proteases and phosphatases, enzymes responsible for breaking down proteins and modifying phosphorylation states, respectively. They are commonly used in various research applications to maintain the integrity and functionality of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence reagent, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Beyotime (1 mentions)
Ab106814, by Abcam (1 mentions)
Ab110304, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Bca kit, by Boster Bio (1 mentions)
Ripa lysis buffer, by Boster Bio (1 mentions)
Hrp conjugated secondary antibody, by Boster Bio (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (1 mentions)
Ripa buffer, by Boster Bio (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
5 protocols using protease and phosphatase inhibitors
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Protein Expression
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Placental tissue samples and HUVECs were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Boster Bio, Pleasanton, CA, USA), and total protein lysates were extracted. The protein concentration was determined using the bicinchoninic acid (BCA) method (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). The proteins were separated using 10% polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with 5% skim milk at room temperature for 2 h, diluted primary antibody was added, and the membrane was incubated at 4 ℃ overnight. Horseradish peroxidase (HRP)-labeled secondary antibody IgG (Abcam, USA) was incubated for 1 h at room temperature. After washing with tris-buffered saline with Tween 20 (TBST), the membrane was color-developed using an ECL luminescent solution (Pierce, Thermo Fisher Scientific, USA). Protein strips were recorded and processed using a Bio-Rad exposure machine (Bio-Rad, Hercules, CA, USA). The relative protein expression was calculated as the ratio of target band to β-actin gray value. The other reagents used were: Primary antibody (Abcam); SIRT1 (ab110304, dilution ratio 1:1,000); PGC-1α (ab106814, dilution ratio 1:1,000); VEGF-A (ab51745, dilution ratio 1:1,000); and β-actin (ab8226, dilution ratio 1:1,000).
3
Western Blot Analysis of Protein Levels
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Proteins were extracted from cells or heart tissues using a RIPA lysis buffer (Beyotime, China) freshly supplemented with protease and phosphatase inhibitors (Boster, China). Bradford assay was used to determine the protein concentrations in the samples (Bio-Rad, USA). Subsequently, the protein samples were electrophoresed in sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel and transferred to PVDF membranes (Millipore, Massachusetts, USA). Membranes were blocked with 5% bovine serum albumin (BSA) (Solarbio, Beijing, China) for 2 h at room temperature. The membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies are listed in Supplementary Table S2 . Afterwards, the membrane was incubated with the secondary antibodies for 1 h at room temperature and visualized by the ECL reagent. The membranes were detected using ChemiDoc™ XRS + system and analyzed using an Image Lab software (Bio-Rad, California, USA). The intensity values of the relative protein levels were normalized to GAPDH.
4
Western Blotting Analysis of JAK-STAT Pathway
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Proteins for western blotting analysis were extracted from the synovial tissues and lysed by RIPA Lysis Buffer (Boster, Wuhan, China) with protease and phosphatase inhibitors (Boster, Wuhan, China). The protein concentration was quantified preliminarily with the BCA kit (Boster, Wuhan, China). The total proteins were separated using 7.5% SDS-PAGE (Epizyme, Shanghai, China), and then transferred onto nitrocellulose membranes. The membranes were blocked by TBST containing 5% skimmed milk for 1 h at room temperature. The primary antibodies including JAK2 polyclonal antibody (1:1,000, Thermo Fisher), phospho-JAK2 polyclonal antibody (1/1,000, Thermo Fisher), JAK3 polyclonal antibody (1:1,000, Thermo Fisher), phospho-JAK3 polyclonal antibody (1:1,000, Thermo Fisher), STAT6 polyclonal antibody (1:1,000, Thermo Fisher), phospho-STAT6 polyclonal antibody (1:1,000, Thermo Fisher) were incubated overnight at 4°C. Next, the membranes were incubated with HRP-conjugated secondary antibody (1/8,000, Boster, Wuhan, China) for 1 h, and then treated with ECL chemiluminescence reagents. Densitometry plots showing protein expression were analyzed by ImageJ (Bethesda, United States). Densitometry plots of the protein expression levels were normalized to GAPDH (1/2,000, Thermo Fisher).
5
Mouse Chondrocyte Protein Extraction and Immunoblotting
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The mouse primary chondrocyte protein extracts were obtained using RIPA Buffer containing protease and phosphatase inhibitors (Boster BIO, China). 20 mg of total protein underwent separation by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), followed by transfer onto PVDF membranes (Millipore, USA) and a 1-h blocking with 5% fat free milk in Tris-buffered saline containing 0.1% Tween-20. Then, successive incubations were performed with rabbit anti-BRAF (1:1000), ERK1/2 (1:1000) p-ERK1/2 (1:1000) and b-actin (1:1000) (Cell Signaling, MA, USA) primary for overnight at 4 C and anti-rabbit secondary (Cell Signaling, MA, USA) for 1 h at ambient antibodies. Enhanced chemiluminescence kit (Bio-Rad, USA) was utilized for detection.
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