Under deep anesthesia with isoflurane, right TGs were collected from mice transcardially perfused with sterile PBS to remove blood and homogenized in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, USA) with protein phosphatase inhibitors (Solarbio, China) and protease inhibitors (Thermo Fisher Scientific, USA) at 1:100. Denaturation of proteins on heating was separated by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, France). Before incubation at 4°C for 6–8 h with primary antibodies (Rabbit Anti-CXCR4 antibody,1:1000, Abcam, USA; Rabbit Anti-CXCL12 antibody, 1:1000, Abcam, USA; Rabbit Anti-β-Tubulin, 1:1000, Proteintech, China), the membranes were blocked with 5% (w/v) bovine serum albumin (Solarbio, China) for 1 h at room temperature. Then, the membranes were cut according to the distribution of the target protein, washed three times and incubated with a corresponding secondary antibody (Goat Anti-Rabbit IgG H&L, 1：1000, Abcam) for 1 h at room temperature. The bands were visualized with western blot detection system (Tanon, China) by High-sig ECL Western Blotting Substrate (Tanon, China) and analyzed with ImageJ Software.
Protein phosphatase inhibitor
Manufactured by Solarbio
Sourced in China, United States
Protein phosphatase inhibitor is a class of chemical compounds that selectively inhibit the activity of protein phosphatases, which are enzymes responsible for the dephosphorylation of proteins. These inhibitors are commonly used in research applications to study the role of protein phosphorylation in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Solarbio (6 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (4 mentions)
Bca protein assay kit, by Solarbio (3 mentions)
Protease inhibitor mixture, by Solarbio (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Wuhan Servicebio Technology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cxcr4 antibody, by Abcam (1 mentions)
Rabbit anti β tubulin, by Proteintech (1 mentions)
Goat anti rabbit igg h l, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Bca protein concentration determination kit, by Solarbio (1 mentions)
β tubulin antibody, by Proteintech (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
19 protocols using protein phosphatase inhibitor
1
CXCR4 and CXCL12 Expression in Mouse Trigeminal Ganglia
2
ANXA1 Protein Expression Analysis
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The RIPA lysate (product number: p00138b) was purchased from Beyotime (Shanghai, China). Protein phosphatase inhibitors and BCA protein concentration determination kits were purchased from Solarbio (Beijing, China). ANXA1 antibody (catalog number: 21990-1-AP) and β-tubulin antibody (catalog number: 10094-1-ap) were obtained from Proteintech (Chicago, IL, United States). The anti-rabbit IgG HRP-conjugated antibody was purchased from Cell Signaling Technology. An ECL chromogenic solution (product number: BL520A) was obtained from Biosharp.
3
Western Blot Analysis of MCM4 Protein
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As previously described [11 (link)], cells were washed with ice-cold PBS twice for 5 min/time and lysed with RIPA (P1300B, Beyotime, Shanghai, China) and protein phosphatase inhibitors (Solarbio). Then, the samples were equilibrated and denatured with 5× loading buffer for 100°C for 10 min. Protein concentrations were measured using BCA kit (PC0020, Solarbio, Shanghai, China). 20 μg of protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane (Millipore), blocked with 3% nonfat milk in TBST (Tris base Tris-buffered saline and 0.1% Triton-100, pH 7.4). Membranes were incubated with primary antibodies for MCM4 (1 : 500, sc-28317, Santa Cruz, USA) and β-actin (1 : 2000, TA-09, ZSGB-BIO, Beijing, China) overnight at 4°C. After washing three times with 1× TBST, they were incubated with secondary antibodies for an hour at room temperature. Again, they were washed three times with 1× TBST. Proteins were visualized by enhanced chemiluminescence (BL520A, Biosharp, Beijing, China) and autoradiography. ImageJ was used to analyze the density of the bands.
4
Western Blot Analysis of Inflammatory Signaling
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Synovial tissues from the right forepaw joints and treated macrophages were lysed in RIPA buffer (Solarbio, Beijing, China, Cat#R0020) with phenylmethylsulfonyl fluoride (Solarbio, Cat#P0100) and protein phosphatase inhibitor (Solarbio, Cat#P1260). Protein samples were separated by SDS-PAGE. Separated proteins were transferred onto polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA, Cat#IPVH00010). For specific protein detection, the following primary antibodies were used: rabbit anti-STAT3 (Cell Signaling Technology, Danvers MA, USA, Cat#9139S), rabbit anti-Phospho-STAT3 (Cell Signaling Technology, Cat#9145S), rabbit anti-NLRP3 (Invitrogen, Waltham, MA, USA, Cat# PA5-79740), mouse anti-HIF-1α (Santa Cruze, Dallas, TX, USA, Cat#13515), and rabbit anti-GAPDH (Abcam, Cambridge, UK, Cat# 8245). Lastly, the Western blotting bands were analyzed with Image J Software.
5
Comprehensive Protein Extraction and Analysis
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Total protein from fresh tissues or cells was lysed using a buffer containing RIPA buffer, 1% protein phosphatase inhibitor (Solarbio, Beijing, China), and 1% phenylmethanesulfonyl fluoride (PMSF, Solarbio, Beijing, China). Protein concentrations were quantified using a BCA Protein assay kit (Solarbio, Beijing, China). Proteins (20 micrograms) were separated on a 4–12% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Merck Millipore) at a current of 300 mA for 95 min. After transfer, the membranes were blocked and incubated with primary antibodies overnight at 4 °C. The primary antibodies used included anti-GAPDH (1:5000, Solarbio), anti-COL-I (1:2000, Abcam), anti-COL-III (1:2000, Abcam), anti- Transforming growth factor -β(TGF-β)(1:3000,Abcam),anti-TN-C (1:2000, Huaxingbio, Beijing), and anti-IL-6 (1:2000, Abcam). This was followed by a 1-hour room temperature incubation with IR Dye-conjugated secondary antibodies (1:5000, ZSGB-BIO, Beijing). The membranes were then imaged and quantified using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). To ensure robustness and reliability of data, the entire experimental procedure was repeated three times.
6
Western Blot Analysis of Protein Phosphorylation
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RAW264.7 cells in 6-well plates (approximately 6 × 105 cells/well) were treated for 15 min at 4 °C in RIPA (#R0010, Solarbio, Beijing, China) lysis buffer with 1 mmol/L protein phosphatase inhibitor (#P1260, Solarbio, Beijing, China) and 1 mmol/L phenylmethanesulfonyl fluoride (#329-98-6, Solarbio, Beijing, China). The lysates were extracted post-centrifuging step (4 °C, 12,000 r/min, 15 min). SDS-PAGE (SDS-polyacrylamide gels, 10%, EpiZyme, Shanghai, China) was used to separate proteins (20 µg per lane) before they were transferred to PVDF (polyvinylidene fluoride, Bio-Rad, Hercules, CA, USA) membranes. The blots were immersed in 5% BSA for 1 h before being incubated with 1:1000 primary antibodies at 4 °C overnight. After that, secondary antibodies with a dilution of 1:5000 were used to probe the blots for 1 h at 37 °C. The ChemiDocXRS + Imaging System (Tanon, Shanghai, China) was used to detect all of the images. Finally, ImageJ (NIH, Bethesda, MD, USA) was used to analyze the protein greyscale values.
7
MHC II and Bcl6 Protein Detection
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The MLNs were cut into pieces and lysed in RIPA buffer (Solarbio, Beijing, China) with protein phosphatase inhibitor (Solarbio), and total proteins were extracted according to the manufacturer’s protocols. Then, the protein concentration was detected by BCA protein assay kit (Solarbio). Each protein sample (50 μg) was electrophoresed on separating 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, MA, United States) at 220 mA for 1.5 h. Afterwards, PVDF membrane was sealed with 5% non-fat powdered milk for 2 h at room temperature and incubated with anti-MHC II antibody (1:500) and anti-Bcl6 (1:250) overnight at 4°C, respectively. The next day, PVDF membrane was washed with 1 × Tris–HCl-buffered saline Tween-20 (TBST) five times for 5 min and incubated with secondary antibodies: HRP-conjugated Affinipure Goat Anti-Mouse or Anti-Rabbit IgG (1,5,000) for 1.5 h at room temperature. Finally, the PVDF membrane was washed and imaged using Amersham Imager 600 chemiluminometer (GE Healthcare Bio-Sciences AB, Sweden). The gray values of protein bands were analyzed by ImageJ software.
8
Ubenimex Impacts Protein Signaling in GC Cells
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Total protein from Ubenimex-treated GC cells was extracted using RIPA lysis buffer containing phenylmethanesulfonyl fluoride (PMSF; Solarbio Biotechnology Co., Shanghai, China) and a protein phosphatase inhibitor (Solarbio) after the cells were washed with ice-cold phosphate-buffered saline (PBS). The protein concentration was measured using the BCA protein assay (Solarbio) after centrifugation at 10,000 rpm for 8 min. Equal quantities of protein were separated on 10% SDS polyacrylamide gels and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were then blocked in 5% nonfat milk for 2 hours at room temperature. The membranes were then incubated overnight at 4 °C with the following primary antibodies: anti-ANPEP, anti-phospho-MAPK, anti-NAB1 and anti-β-actin. The membranes were then washed three times with Tris-buffered saline-Tween (TBST, Solarbio) and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1.5 hours. The data were analyzed using Bio-Rad Quantity One Software v4.62 (Bio-Rad Laboratories Co., Ltd.). All Western blot analyses were performed at least three times. The grayscale values of the target bands were analyzed using ImageJ software (NIH Image, Bethesda, MD, USA).
9
Apoptosis and Cell Cycle Regulation
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Cells were treated with the above concentrations of FB1 for 48 h, then washed three times with ice-cold PBS. The cells were then lysed with RIPA buffer (Solarbio) containing a protease inhibitor mixture (Solarbio, Beijing, China) and protein phosphatase inhibitor (Solarbio). The total protein concentrations were determined using a Bradford protein kit (Tiandz, Beijing, China). The total proteins were resolved by electrophoresis and transferred to poly(vinylidene fluoride) membranes for 80 min at 200 mA. PVDF membranes were incubated with primary antibodies caspase3 (Beijing Biosynthesis Biotechnology Co., Ltd. Beijing, China), caspase9 (Bioss), CDK2 (Bioss), CDK4 (Bioss, Beijing, China), cyclinD1 (immunoway, Plano, TX 75024, USA), cyclinE1 (immunoway), Bax (Proteintech Group, Inc., Wuhan, China), and GAPDH (Servicebio, Wuhan, China) overnight at 4 °C. Then, the membrane was washed three times with Tris-buffered saline tween (TBST) for 15 min. Secondary antibody (IgG, Vazyme) was used to incubate the membranes for 1 h at 4 °C. Finally, band density was quantified using the Image Lab 5.0 on the ChemiDOC XRS + system (Bio-Rad, Hercules, CA, USA).
10
Western Blot Analysis of Focal Adhesion Proteins
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Cells were lysed in RIPA buffer (Beyotime) containing protease inhibitor PMSF (Solarbio) at ratio (100:1 = RIPA: PMSF) and protein phosphatase inhibitor (Solarbio), then clarified by centrifugation at 13,000 g for 10 min at 4°C. Protein concentration was quantified using BCA Protein Assay Kit (Solarbio, Beijing, China). Western blot was performed by automatic protein analyser (Wes&Jess) according to the manufacturer's instruction. All protein levels were normalized to β‐actin. The blots were reacted with rabbit anti‐human against β‐actin (CST), phospho‐FAK, FAK, phospho‐MEK1, MEK1, phospho‐ERK1/2, ERK1/2, β3, αv, α5, β1 (all from Abcam).
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