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.
Protease inhibitor
Manufactured by MedChemExpress
Sourced in China, United States
Protease inhibitors are compounds that block the activity of proteases, which are enzymes responsible for the breakdown of proteins. These inhibitors are commonly used in various research and therapeutic applications, such as the study of protein function and the development of drugs for conditions like viral infections, cancer, and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by MedChemExpress (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Ripa lysis buffer, by Solarbio (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Hy k0010, by MedChemExpress (2 mentions)
Rnase inhibitor, by Merck Group (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Pr 619, by MedChemExpress (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Nh4cl, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence agent, by Merck Group (1 mentions)
Ripa buffer, by MedChemExpress (1 mentions)
Immobilon ecl substrate, by Merck Group (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Methanol activated polyvinylidene fluoride membrane, by Merck Group (1 mentions)
21 protocols using protease inhibitor
1
Preparation of UP-nanovaccine from Cell Lysate
2
Western Blot Analysis of Lung Tissue
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The middle lobe of the right lung tissue was lysed with RIPA lysis buffer (Solarbio, Beijing, China) and protease inhibitors (MedChemExpress, NJ, USA). Tissue lysates were centrifuged at 13,000 g for 15 min at 4 °C. The protein concentration was measured using a BCA protein assay kit (Pierce, CA, USA). The protein sample (40 μg) was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to PVDF membranes (Millipore, Darmstadt, Germany), and then incubated with diluted primary antibodies (1:1000) overnight at 4 °C. After incubation with the secondary antibody, target proteins were detected with an enhanced chemiluminescence agent (Millipore, MA, USA). β-actin served as an internal control.
3
Western Blot Protein Quantification Protocol
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HASMCs and tissue sample proteins were extracted using RIPA buffer containing protease inhibitors (MedChemExpress, NJ, USA). The protein concentration was measured using BCA Protein Assay Reagent (Thermo Fisher Scientific). Lysates were denatured by boiling with SDS-PAGE Buffer loading buffer (Reducing, 5×) (CWBIO, Beijing, China). The proteins and prestained protein ladder (Thermo Fisher Scientific) were resolved on 10% SDS-PAGE gels and transferred to methanol-activated polyvinylidene fluoride membranes (Millipore, MA, USA). Membranes were blocked with 5% nonfat dry milk for regular antibodies, and incubated with primary antibodies overnight at 4 °C. The next day. the membranes were incubated with HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Detection was performed with Immobilon ECL substrate (Millipore), and the blots were imaged with a ChemiDoc Touch Imaging System (Bio-Rad). Protein expression was quantified using Image Lab 3.0 (Bio-Rad); target protein expression was normalized to that of GAPDH or β-actin in each sample and is expressed as a percentage of the control. The primary antibodies used in the experiments are shown in Table S2 .
4
Western Blot Protein Detection Protocol
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Tissues or cells were homogenized in a RIPA lysis buffer (150 mM NaCl, 50 mM Tris HCl [pH 7.4], 2 mM EDTA, 0.1% SDS, 1% NP-40) supplemented with protease inhibitors (Catalog# HY-K0010, MedChemExpress) and phosphatase inhibitor cocktail (Catalog# B15001, Bimake). Proteins were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Catalog# 1620177, BIO-RAD). The membrane was blocked with 10% nonfat milk for an hour, followed by incubation with primary antibody at 4 °C overnight. The membrane was washed with TBST (2.7 mM Tris base, 137 mM NaCl and 0.1% Tween 20) and then incubated with corresponding secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology) for 1 h at room temperature. Details of primary and secondary antibodies are listed in Supplementary Table 3 . After washing with TBST, the proteins were visualized using enhanced chemiluminescence reagents (BIO-RAD) and quantified using ImageJ.
5
Quantifying Histone Lysine Lactylation
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Total proteins in cells were extracted using RIPA buffer (Aspen Biological, Wuhan, China) and protease inhibitors (MedChemExpress, SNJ, USA). Protein concentration was measured using the BCA kit (Aspen). Equal amounts of protein were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocked with milk at room temperature for 1 h. PVDF membranes were washed three times with TBST for 10 min each, and then incubated overnight at 4 °C with the appropriate primary antibody. Following another round of washing, the membranes were incubated with secondary antibodies at room temperature for 1 h. Finally, the membranes were developed using ChemiDoc XRS gel imaging system (Bio-Rad, Hercules, CA, USA). The antibodies used in this experiment are listed below: Anti-beta Tubulin (PTM-6414, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys18) (PTM-1427RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys14) (PTM-1414RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys9) (PTM-1419RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl Lysine (PTM-1425, PTMBIO, Zhejiang, China); Anti-Histone H3 (PTM-6621, PTMBIO, Zhejiang, China); SLC25A29 (26663-1-AP, Proteintech, Wuhan, China).
6
Detecting MDR1 and Ubiquitin in Tumor Cells
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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.
7
Protein Extraction and Western Blot Analysis
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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).
8
Investigating NF-κB Signaling and Osteogenesis
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The RAW 264.7 cells were cultured with PBS, LPS, KR−1, and KR−2 media for 30 min, and MC3T3-E1 cells were grown in various CM osteogenic media for 14 days. Subsequently, the cells were lysed for 30 min in lysis buffer with protease inhibitors (MedChemExpress), and cell lysates were analyzed by gel electrophoresis after samples were collected for processing and transferred to nitrocellulose membranes. The membrane was incubated with the relevant antibody for 12 h at 4 °C before exposure, after blocking had been performed with a 5% bovine serum albumin solution (Solarbio) for 1 h. The following antibodies were used for Western blotting: p65, phospho-p65, IκBα, phospho-IκBα, GAPDH, β-tubulin, OCN, Runx-2, and COL1α1 (all 1:1000 dilutions, Abcam, Cambridge, UK).
9
Purification of Polyvalent Capsids
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The purification steps of PVC can be found in previous literature with minor modifications (12 (link)). Briefly, the LysR-producing plasmid pBR60 was transformed into E. coli EPI300 strain harboring plasmid pCNM3, which expresses PVC structural genes. If cargo protein was included, a third pBBRN plasmid expressing relevant genes was transformed into the strain as well. Overnight cultures were inoculated into 200 ml of LB broth for another 16-hour growth at 30°C. Bacterial pellets were collected and lysed in 30 ml of buffer P [25 mM tris (pH 7.4), 140 mM NaCl, 3 mM KCl, deoxyribonuclease I (50 μg ml−1), lysozyme (200 μg ml−1), 0.5% Triton X-100, 5 mM MgCl2, and 1× protease inhibitor (MedChemExpress)] for 30 min at 37°C. After centrifugation of cell lysates (14,000 rpm, 10 min), the supernatant was ultracentrifuged at 150,000g for 60 min at 4°C. The pellet was suspended in 1 ml of sterile phosphate-buffered saline (PBS) after centrifugation. After another centrifugation at 14,000 rpm for 10 min at 4°C, the supernatant was ultracentrifuged for the second time at 150,000g for 60 min at 4°C to pellet the protein complex. The pellet was resuspended in 200 μl of ice-cold PBS and centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant containing the PVC complex was stored at 4°C for short-term usage.
10
Protein Extraction and Western Blotting
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The cells were lysed in RIPA buffer (Macgene, Beijing, China, #MP015) supplemented with a protease inhibitor (MedChemExpress, Monmouth Junction, NJ, USA, HY-K0012). After determining the protein concentration using a BCA Protein Assay Kit (Thermo Fisher Scientific, Bremen, Germany, #23227), equivalent protein quantities were subjected to SDS–PAGE and then transferred to PVDF membranes (Millipore, Darmstadt, Germany, #GVWP02500). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then probed with the indicated primary antibodies, followed by the appropriate HRP-conjugated anti-mouse/rabbit secondary antibodies (Zsgb, Beijing, China, #ZB-2301). Immunoreactive bands were measured with an enhanced chemiluminescence western blotting system (Millipore, Darmstadt, Germany, #WBKLS0500). For the detection of ATX, a secreted protein, cells were cultured in serum-free medium. The medium was concentrated (20-fold) using an Amicon Ultra 30000 (Merck KGaA, Darmstadt, Germany), and then subjected to SDS-PAGE and western blotting.
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