Vx3 protein was obtained and covalently linked to α-Al2O3 nanoparticles to generate α-Al2O3-Vx3 nanoparticles according to our previous reports (12 (link), 13 (link)). 4T1/WT cells and 4T1/EPB cells were treated with 200 nM bortezomib (Millennium Pharmaceuticals, USA) and 20 mM NH4Cl (Sigma, USA) for 9 h. The cells were collected and lysed in RIPA lysis buffer (Millipore, USA) containing protease inhibitors (MedChemExpress, USA), phosphatase inhibitors (MedChemExpress, USA), and PR-619 (MedChemExpress, USA). The cell lysate was reacted with α-Al2O3-Vx3 nanoparticles under stirring for 12 h at 4°C to generate α-Al2O3-UPs nanovaccine (named UP-nanovaccine) according to our previous study (13 (link), 20 (link)). The precipitates (UP-nanovaccine) were collected by centrifugation (12,000 g, 30 min, 4°C), and the supernatant was collected as unbound lysate, followed by the detection of ubiquitin protein levels in the three samples using Western blotting. The covalently linked product UP-nanovaccine was collected by centrifugation, and the number of UPs enriched by α-Al2O3-Vx3 was evaluated by collecting and calculating the difference between the number of UPs in the supernatant before and after the reaction by BCA Protein Assay Kit (Beyotime Biotechnology, China) according to the protocol of the manufacturer.
Phosphatase inhibitor
Manufactured by MedChemExpress
Sourced in United States, China
Phosphatase inhibitor is a chemical compound used in laboratory settings to inhibit the activity of phosphatase enzymes. Phosphatases are responsible for the removal of phosphate groups from various biomolecules, and inhibiting their activity can be useful in a variety of experimental and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by MedChemExpress (6 mentions)
Proteinase inhibitor, by MedChemExpress (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor, by Beyotime (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Enhanced chemiluminescence kit, by Vazyme (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Bca protein assay kit, by Vazyme (2 mentions)
Anti erk1 2, by Proteintech (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Gapdh, by BioLegend (2 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Nh4cl, by Merck Group (1 mentions)
Pr 619, by MedChemExpress (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Anti cdk7, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
13 protocols using phosphatase inhibitor
1
Preparation of UP-nanovaccine from Cell Lysate
2
Western Blot Analysis of Cell Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted using RIPA buffer (Sangon Biotech, Shanghai, People’s Republic of China) containing proteinase inhibitors (MedChem Express) and phosphatase inhibitors (MedChem Express). The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). The following antibodies were used for Western blot: anti-CDK7 (Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (Cell Signaling Technology), anti-poly(ADP-ribose) polymerase (anti-PARP) (Cell Signaling Technology), anti-cleaved PARP (Cell Signaling Technology), anti-RNA Pol II (Abcam, Cambridge, UK), anti-phospho RNA Pol II (Ser2) (Abcam), anti-phospho RNA Pol II (Ser5) (Abcam), anti-phospho RNA Pol II (Ser7) (Millipore), anti-CDK1 (Abcam), and anti-CDK1 (phospho T161) (Abcam).
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cancer cells were rinsed two times with 1 mL of cold PBS supplemented with 1% Na3VO4 and then scraped into a lysis buffer containing a mixture of proteinase (Sigma-Aldrich, Saint Louis, MO, USA) and phosphatase inhibitors (MedChem Express, Monmouth Junction, NJ, USA) [31 (link)]. Next, the cell lysates were centrifuged at 4 °C and 13,000× g for 10 min to obtain the supernatant as the cellular proteins or extracts. Protein concentrations were determined using a Bradford assay (Bio-Rad, Hercules, CA, USA). For Western blotting, 30 μg cell extracts were separated through 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes to detect the indicated molecules, with β-actin used as the loading control. The Western blotting assay was performed as described previously [32 (link)].
4
Detecting MDR1 and Ubiquitin in Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tumor cells were lysed in RIPA lysis buffer containing protease inhibitors (MedChemExpress, USA) and phosphatase inhibitors (MedChemExpress, USA), and MDR1 was determined using anti-MDR1 (1:1,000, Abcam, ab170904, UK) as the primary antibody and goat anti-rabbit IgG HRP as the secondary antibody (1:5,000, eBioscience, USA). Actin served as the endogenous control.
For ubiquitin detection, an equal amount of protein (20 µg) was loaded from all samples, as assessed by Coomassie blue-stained SDS-PAGE. Anti-ubiquitin antibody (1:1,000, Sigma, #3933, USA) served as the primary antibody, and goat anti-rabbit IgG HRP (1:5,000, eBioscience, USA) served as the secondary antibody.
For ubiquitin detection, an equal amount of protein (20 µg) was loaded from all samples, as assessed by Coomassie blue-stained SDS-PAGE. Anti-ubiquitin antibody (1:1,000, Sigma, #3933, USA) served as the primary antibody, and goat anti-rabbit IgG HRP (1:5,000, eBioscience, USA) served as the secondary antibody.
5
Western Blot Analysis of Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor tissue and cell lysates were prepared using RIPA lysis buffer from Beyotime Biotechnology in China, along with PMSF, protease inhibitor cocktail, and phosphatase inhibitors from MedChemExpress. Protein quantification for tumor tissues and cells was performed with the BCA kit from TransGen Biotech in China. Following gel electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA for 1 h at room temperature. Afterward, they were incubated overnight at 4 °C with primary antibodies (Table S4 , Supporting Information), followed by incubation with HRP‐conjugated secondary antibodies (ab205718 and ab205719, Abcam, 1:5000) in accordance with the manufacturer's protocol.
6
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues or cells were resuspended in the cool radio immunoprecipitation assay (RIPA) lyris buffer (Beyotime, Shanghai, China) containing 1 % protease inhibitors (MedChemExpress) and 1 % phosphatase inhibitors (MedChemExpress) for 30 min on ice. Then samples were centrifuged at 12,000 rpm for 20 min at 4 °C and the supernatant containing protein was removed. Total protein concrentration was determined with use of the BCA kit (Solarbio) and protein samples were then heated within 5 × loading buffer (Beyotime) at 99.5 °C for 10 min. Equal amounts of protein were fractionated using 8 %, 10 %, 12 % or 15 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5 % skim milk for 1 h at room temperature and incubated overnight at 4 °C with the primary antibodies. Information regarding primary antibodies is available in Table S3 . After an overnight incubation, membranes were incubated with the secondary antibodies (1:5000, Solelybio, Beijing, China) for 1 h at room temperature. Bands were detected using chemiluminescence (ECL) HRP substrate (Merck Millipore) under an chemiluminescence imager (Tanon4800, Shanghai, China).
7
Analysis of Adipose Tissue MAPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse adipose tissue was homogenized with a tissue homogenizer at centrifuge of 12000 g at 4 °C for 10 min using RIPA lysis buffer containing protease inhibitor (Beyotime Biotechnology) and phosphatase inhibitor (MedChemExpress).The supernatant was aspirated, and the protein concentration was measured by the BCA Protein Assay Kit (Vazyme Biotech Co., Ltd.).The protein sample was loaded for polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane in an electrophoresis transfer buffer containing 10% methanol. The membrane was blocked with 5% BSA (Sigma-Aldrich) for 2 h at room temperature, followed by incubation overnight at 4 °C with anti-p38(1:500; Proteintech™), anti-phosphorylation p38(1:1000;Beyotime), anti-ERK1/2(1:2000; Proteintech), anti-phosphorylation ERK1/2(1:1000; Beyotime), anti-JNK(1:3000; Proteintech™), anti-phosphorylation JNK (1:1000; Beyotime), and anti-GAPDH (1:50,000; Proteintech Wuhan, China) antibodies. After overnight incubation, the blots were incubated with the corresponding secondary antibodies (1:10,000; Bioworld) for 1 h at room temperature. Bands of target proteins were visualized using an enhanced chemiluminescence kit (Vazyme Biotech Co.,Ltd).
8
Western Blot Analysis Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in 10 cm dishes to 90% confluence and then harvested and lysed with RIPA lysis buffer (lot: R0010, Solarbio, Inc., Beijing, CHN) containing protease inhibitor (MedChemExpress, New Jersey, USA) and phosphatase inhibitor (MedChemExpress, New Jersey, USA). Then, proteins were separated by SDS–PAGE, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Finally, membranes were blocked and blotted with the corresponding antibodies and detected by enhanced chemiluminescence reagent (Thermo Fisher Scientific, MA, USA) with C300 (Azure Biosystems, CA, USA).
9
Protein Extraction and Immunoblotting Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytoplasmic and nuclear proteins were extracted by NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific™) with proteinase inhibitor (MedChemExpress, Monmouth Junction, NJ, USA) and phosphatase inhibitor (MedChemExpress) [44 (link)]. The cell lysates were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with the following indicated antibodies: bone morphogenetic protein-4 (BMP4; Cusabio Life Science, Wuhan, China), runx family transcription factor 2 (Runx2; Novus Biologicals, Centennial, Colorado, USA), ALP (R&D Systems, Minneapolis, MN, USA), extracellular signal-regulated kinase (ERK; Cell Signaling Technology), phospho-ERK (p-ERK; Cell Signaling Technology), c-Jun N-terminal kinase (JNK; Cell Signaling Technology), p-JNK (Cell Signaling Technology), p38 (Cell Signaling Technology), p-p38 (Cell Signaling Technology), β-catenin (Cell Signaling Technology), Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (BioLegend, SanDiego, CA, USA). GAPDH was used as a housekeeping gene for normalization. Lamin B1 was used for normalization of nuclear β-catenin.
10
Western Blot Analysis of PI3K/AKT Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with lysate buffer containing a protease inhibitor (Beyotime Biotechnology) and phosphatase inhibitor (MedChemExpress) in accordance with the manufacturers’ instructions. After extracting proteins, a BCA Protein Assay Kit (Vazyme Biotech Co., Ltd.) was used to measure the concentration. The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore). After the membranes were blocked in 5% BSA (Sigma-Aldrich) for 2 h, primary antibodies were applied at 4 °C overnight on a horizontal shaker. The next day, the secondary antibody (1:10,000; Bioworld) was applied for 1 h at room temperature. The primary antibodies were anti-GAPDH (1:50,000; Proteintech Wuhan, China), anti-PI3K p85 (1:1000, 4257, CST), anti-PI3K p85 Y458 (1:1000, 4228, CST), anti-AKT (1:1000; ab179463, Abcam), anti-phosphorylation AKT (1:1000; ab81283, Abcam), anti-GSK3β (1:2000; ab32391, Abcam), anti-phosphorylation GSK3β S9 (1:1000; ab75814, Abcam), anti-ERK1/2 (1:2000; Proteintech, and anti-phosphorylated ERK1/2(1:1000; Beyotime). PI3K inhibitor LY294002 was purchased from MedChemExpress (HY-10108). Visualization of the protein bands was conducted with an enhanced chemiluminescence kit (Vazyme Biotech Co.,Ltd).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!