Treated cells were lysed with IP lysis buffer (Beyotime Biotechnology) containing Protease/Phosphatase Inhibitor Cocktail (Beyotime Biotechnology). Cell lysates were centrifuged, and the supernatants were collected and incubated with specific antibodies or normal IgG overnight at 4°C. Next day, Protein A/G Beads (Santa Cruz Biotechnology) were added into the supernatants and incubated for 3 h at 4°C. After washing protein‐binding beads, the co‐precipitated proteins were subjected to western blot assays as described above.
Protease phosphatase inhibitor cocktail
Manufactured by Beyotime
Sourced in China
Protease/phosphatase inhibitor cocktail is a laboratory reagent designed to protect protein samples from degradation by proteases and dephosphorylation by phosphatases. It is a mixture of various chemical inhibitors that work together to preserve the integrity of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Protein a g bead, by Santa Cruz Biotechnology (1 mentions)
Ip lysis buffer, by Beyotime (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
β actin, by Beyotime (1 mentions)
Novex nupage gel system, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence substrate, by Roche (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Lipofectamine 3000, by Merck Group (1 mentions)
Bca protein assay kit, by Ipsen (1 mentions)
Anti ccr7, by R&D Systems (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Sds page loading buffer, by Beyotime (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
St506, by Beyotime (1 mentions)
12 protocols using protease phosphatase inhibitor cocktail
1
Immunoprecipitation Assay Protocol
2
TMEM127 Protein Expression Quantification
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Protein samples were collected using lysis buffer containing a protease/phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Proteins were separated using SDS-PAGE and transferred to PVDF membranes (BioRad, AL, USA). After blocking with western blot blocking solution for 1 h, TMEM127 (Immunoway, USA) and β-actin(Dawen, HangZhou, China) primary antibody were incubated overnight. The next day the membrane was incubated with the secondary antibody for 2 h. Proteins were detected using a chemiluminescence kit (Sangson Biotech, Shanghai, China) according to the manufacturer’s instructions. Each experiment was performed in triplicate.
3
Protein Extraction and Western Blot Analysis
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Briefly, isolation of total protein from liver tissues was performed using RIPA Lysis Buffer (Beyotime, Shanghai, China) added with Protease-Phosphatase Inhibitor Cocktail (Beyotime, Shanghai, China) on ice, and the supernatants were collected after centrifugation. Protein concentrations were measured using a BCA Protein Assay kit (Beyotime, Shanghai, China). Equal proteins of each group were loaded on 10% SDS-PAGE to separate them and were delivered into polyvinylidene difluoride membrane. The membranes were blocked in 5% skimmed milk, which dissolved in Tris buffered saline with Tween 20. Then, they were incubated with primary antibodies against Cleaved-GSDMD (10,137; 1:1000; CST), β-actin (AF5003; 1:5000; Beyotime) overnight at 4 °C. After being incubated with secondary antibodies (A0208; 1:1000; Beyotime), the blots were visualized with enhanced chemiluminescence BeyoECL Star (Beyotime, Shanghai, China), and the chemiluminescent signals were captured using the ChemiDoC XRS+ system. Finally, the relative intensity of the chemiluminescent signals was quantified using the ImageJ software.
4
Muscle Protein Extraction and Analysis
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Total protein was extracted from muscle tissue in RIPA lysis buffer supplemented with a protease/phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Protein concentration was measured using a BCA assay. Lysates of 20 μg were then subjected to electrophoresis using the NuPAGE Novex Gel system (Thermo Fisher Scientific, Waltham, MA) for SDS-PAGE. Anti MMPs antibody, anti-GAPDH antibody, and HRP-coupled secondary antibodies were used. Signals were visualized with chemiluminescent substrate and densitometric analysis was performed with Image-pro plus.
5
Western Blot Protein Analysis Protocol
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The cells were lysed on ice for 10 min with RIPA lysis buffer (P0013C, Beyotime, Shanghai, China) containing 1× Protease Phosphatase Inhibitor Cocktail (P1050, Beyotime) before being centrifuged at 13,000 g for 10 min at 4 °C. Total protein samples (40 µg) were resolved on 8% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk in PBST (0.05% Tween 100 in PBS), incubated with specified primary antibodies overnight at 4 °C, washed and incubated with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA), and then washed and detected with an enhanced chemiluminescence substrate (Roche, Indianapolis, IN, USA).
6
Investigating Protein Interactions and Signaling Pathways
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Cells were lysed in the RIPA buffer (Beyotime) containing 1% Protease/Phosphatase Inhibitor Cocktail (MCE) and PMSF. The BCA Protein Assay Kit (Epizyme) was applied to measure protein concentrations. Western blot analysis was carried out as previously described.22 For coimmunoprecipitation assays, MIA‐PaCa‐2 and SW1990 cells were transfected with the indicated plasmids according to manual of Lipofectamine 3000’s manufacturer (Sigma‐Aldrich). Immunoprecipitation was undertaken as described previously.22 , 23 In this study, anti‐PD‐L1 (ab228415) was purchased from Abcam. Anti‐phospho‐AKT (Ser473)(9271), anti‐AKT (9272), anti‐phospho‐GSK‐3β (ser9)(9323), anti‐GSK‐3β (12456), anti‐GAPDH (5174), and anti‐β‐actin (4970) were purchased from Cell Signaling Technology. Anti‐Ccl21 (AF457‐SP) and anti‐CCR7 (150503) were purchased from R&D Systems.
7
Western Blot Protein Analysis
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The cells were washed 3 times with cold phosphate-buffered saline (PBS) and incubated on ice with radioimmunoprecipitation assay (RIPA) buffer (Beyotime) containing a protease/phosphatase inhibitor cocktail (Beyotime) and phenylmethylsulfonyl fluoride (PMSF) (Beyotime) for 30 min. The lysates were centrifuged for 10 min at 4°C. Equal amounts of protein were denatured for 5 min in 5× SDS-PAGE loading buffer (Beyotime). The samples were electrophoresed and electrotransferred onto nitrocellulose membranes. Subsequently, the membranes were incubated with 5% nonfat dry milk for 2 h at room temperature and incubated with primary antibody and then secondary antibody. The membranes were visualized with an Odyssey system. Data from one experiment of three are shown. The target protein relative intensity, which was relative to GAPDH expression, was quantified using Image Studio V5.2.
8
Phosphorylation Signaling Pathway Analysis
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Human washed platelets preincubated with PD alone or combined with VP at 37°C for 15 min were lysed by 100 μL RIPA buffer containing protease/phosphatase inhibitor cocktail (Beyotime Biotechnology). 30 μg proteins were separated by 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane for 1 h. After blocking, the specific antibodies against phospho-Akt, Akt, phospho-PI3K, PI3K, phospho-GSK3β, GSK3β, and β-actin were incubated overnight at 4°C, and secondary antibodies were kept at room temperature for 1 h. After washing, the protein band was visualized and analyzed using FluorChem HD2 (California, USA).
9
Quantification of Protein Expression
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On day 3 post-transfection, total protein was extracted from the transfected cells using cell lysis buffer (P0013, Beyotime) with PMSF (ST506, Beyotime) and protease phosphatase inhibitor cocktail (P1046, Beyotime), followed by BCA assay for protein quantification (P001, Beyotime). Protein lysates in InstantView™ SEMS-PAGE protein staining and loading buffer (Beyotime, P0280) were loaded on 10% sodium dodecyl sulfate-polyacrylamide (SEMS-PAGE) gels and then transferred to polyvinylidene difluoride (PVDF) membranes (cat#1620177, Bio-Rad), blocked with 5% skimmed milk, and incubated with primary antibodies for CEP192 (1:1,000; Bethyl, A302–324A) and GAPDH (1:1,000; #8884, Cell Signaling Technology) overnight at 4°C. The membranes were washed and incubated with horse anti-rabbit IgG coupled with horseradish peroxidase (HRP) (1:10,000; #7076, Cell Signaling Technology) for 1 h, then developed with SuperSignal TM West Pico PLUS Chemiluminescent Substrate (WB322159, Thermo). Images were acquired with ChemisScope 6100 Touch (Clinx, China).
10
Protein Extraction and Signaling Analysis
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Protein was extracted from livers using RIPA lysis buffer containing protease/phosphatase inhibitor cocktail (Beyotime Biotechnology). Antibodies against p-AKT (phospho Ser473, ab66138, Abcam), AKT (#4691, Cell Signaling Technology), p-GSK3β (phospho Ser9, #9322, Cell Signaling Technology), and GSK3β (#12456, Cell Signaling Technology) were used.
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