Carotid arteries and VSMCs were extracted, lysed with RIPA buffer (Beyotime, Shanghai, China), and then subjected to western blotting analysis as described in our previous research [5 (link)]. VSMC extracts were prepared in lysis buffer (50 mmol/L Tris·HCl (pH 7.5), 150 mmol/L NaCl, 0.5% IGEPAL CA-630, 0.5% deoxycholic acid, 0.1% SDS, 1 mmol/L EDTA, 1 mmol/L NaF, 0.1 mmol/L Na3VO4, 50 μmol/L phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, 5 μg/mL aprotinin; Solarbio, Beijing, China) for immunoprecipitation assay. Samples were incubated with the primary antibody at 4°C overnight, and immunocomplexes were precipitated after 1 h of incubation with sepharose A/G beads (Santa Cruz, Dallas, USA). Antibodies against PGC-1α (cat ab106814) and GAPDH (cat 2118S) were purchased from Abcam and Cell Signaling Technology, respectively. Antibody against Plin5 was purchased from Novus (cat NB110-60,509).
Leupeptin
Manufactured by Solarbio
Sourced in China
Leupeptin is a protease inhibitor that targets a broad range of serine and cysteine proteases. It is commonly used in laboratory research to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Aprotinin, by Solarbio (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Sepharose a g beads, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Methyl jasmonate, by Merck Group (1 mentions)
L phenylalanine, by Solarbio (1 mentions)
Methanol, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Tianjin Kemiou Chemical Reagent (1 mentions)
Hydrogen peroxide, by Tianjin Kemiou Chemical Reagent (1 mentions)
Sodium dihydrogen phosphate, by Tianjin Kemiou Chemical Reagent (1 mentions)
Folin ciocalteu reagent, by Solarbio (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
Ethanol, by Tianjin Kemiou Chemical Reagent (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
β glycerophosphate, by Solarbio (1 mentions)
Elisa reagent kits, by R&D Systems (1 mentions)
7 protocols using leupeptin
1
VSMC Protein Extraction and Analysis
2
Antioxidant Profiling of Plant Compounds
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Ethanol, sodium carbonate (Na2CO3), Disodium hydrogenorthophosphate, sodium dihydrogen phosphate, 30% hydrogen peroxide, Vit C, catechol, 2-methoxyphenol, β-mercaptoEthanol, polyvinylpyrrolidone (PVPP), glycerol, tris-base, hydrochloric acid, and magnesium chloride (MgCl2) were obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Folin–Ciocalteu reagent, potassium persulfate, leupeptin, phenylmethylsulfonyl fluoride, boracic acid, borax, and l -phenylalanine were purchased from Beijing Solarbio Science and Technology CO., Ltd. (Beijing, China). Gallic acid, hydroxybenzoic acid, chlorogenic acid, caffeic acid, sinapic acid, ferulic acid, rutin, cinnamic acid, catechin, and quercetin, were obtained from Shanghai Yuanye Biochemical Co., Ltd. (Shanghai, China). 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) and adenosine triphosphate were obtained from Shanghai Aladdin Biochemical Co., Ltd. (Shanghai, China). Methyl jasmonate and mEthanol were obtained from Sigma Chemical Co. (St. Louis, MO, USA). MEthanol was reagent of HPLC-grade. The other chemical reagents were of reagent grade.
3
STING Pathway Activation Analysis
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Activation of the STING pathway was assessed by western blot to analyze phosphorylation status of STING, TBK1, and IRF3 using commercially available antibodies. Cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1% Sodium Deoxycholate, 1 mM Na3VO4, 25 mM β-glycerol-phosphate) supplemented with 0.1 mM PMSF (Beyotime Biotechnology) and 0.5 mg/ml Leupeptin (Solarbio) on ice for 30 min. Lysates were centrifuged at 13,000 rpm for 15 min at 4°C, and the soluble fraction was transferred to a new tube. Protein concentration was determined by the absorbance of 280 nm on NanoDrop One (Thermo Fisher Scientific), and the protein sample was boiled with SDS loading buffer at 95°C for 5 min. Proteins were separated on 12% SDS-PAGE gels, immunoblotted onto nitrocellulose membranes, and subsequently incubated with different primary antibodies overnight. After incubation with HRP-labelled secondary antibodies (Huaxingbio) for 1 h at room temperature, the proteins were detected using ECL substrates (Mei5 Biotechnology).
4
Protein Expression Analysis in Rat Arteries
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Vascular smooth muscle cells or rat arteries were lysed for 30 minutes on ice in lysis buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, 2 mmol/L sodium pyrophosphate, 25 mmol/L β‐glycerophosphate, 1 mmol/L EDTA, 1 mmol/L Na3VO4, 0.5 μg/mL leupeptin and 0.1 mmol/L PMSF (R0010) from Solarbio. The supernatant was collected after centrifugation at 12 000 g for 10 minutes at 4°C.
Proteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes from Millipore. After blocking in 5% nonfat milk for 1 hour at room temperature, the PVDF membranes were probed with primary antibodies against α‐SMA (1:1000 dilution), calponin 1 (1:1000 dilution), RUNX2 (1:500 dilution), β‐catenin (1:500 dilution), phospho‐β‐catenin (Ser675) (1:1000 dilution), GSK‐3β (1:1000 dilution), phospho‐GSK‐3β (Ser9) (1:1000 dilution), PPAR‐γ (1:1000 dilution), histone‐H3 (1:1000 dilution) and GAPDH (1:1000 dilution) overnight. The membranes were then washed with TBS‐T, followed by an incubation with a horseradish peroxidase‐conjugated secondary antibody (1:8000 dilution) (ZSGB‐BIO) for 1.5 hours at room temperature; then, the membranes were developed with chemiluminescence and were stripped and reprobed when necessary.
Proteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes from Millipore. After blocking in 5% nonfat milk for 1 hour at room temperature, the PVDF membranes were probed with primary antibodies against α‐SMA (1:1000 dilution), calponin 1 (1:1000 dilution), RUNX2 (1:500 dilution), β‐catenin (1:500 dilution), phospho‐β‐catenin (Ser675) (1:1000 dilution), GSK‐3β (1:1000 dilution), phospho‐GSK‐3β (Ser9) (1:1000 dilution), PPAR‐γ (1:1000 dilution), histone‐H3 (1:1000 dilution) and GAPDH (1:1000 dilution) overnight. The membranes were then washed with TBS‐T, followed by an incubation with a horseradish peroxidase‐conjugated secondary antibody (1:8000 dilution) (ZSGB‐BIO) for 1.5 hours at room temperature; then, the membranes were developed with chemiluminescence and were stripped and reprobed when necessary.
5
Lung Cytokine Quantification Protocol
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After the mice were sacrificed, the lungs were harvested, and of tissue per animal was homogenized in PBS containing 1% (wt/vol) Triton X-100 (Beijing Solarbio Technology Co., Ltd.) and pepstatin A, leupeptin, and aprotinin (all at , pH 7.4; Beijing Solarbio Technology Co., Ltd.) and incubated on ice for 30 min as previously described (Wieland et al. 2006 (link)). The homogenate was then centrifuged (4°C, , 15 min), and the supernatant was assayed for IL-4, IL-13, and interferon using the appropriate ELISA reagent kits (R&D Systems, Inc.) according to the manufacturer’s instructions.
6
Investigating NLRP3 Inhibition in Atherosclerosis
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MCC950, a small molecule NLPR3 inhibitor, and olaparib were purchased from Selleck Chemicals (Houston, TX); oxLDL was purchased from Anhui Yiyuan Bio-technology (Bozhou, China); HyClone RPMI 1640 and DMEM were purchased from Cytiva (Shanghai, China); fetal bovine serum (FBS) and MitoSox Red were purchased from Thermo Fisher Scientific (Beijing, China); aprotinin and leupeptin were purchased from Solarbio Life Science (Beijing, China); DTT (dithiothreitol) was purchased from Wako Pure Chemical Industries (Osaka, Japan); protein A/G-agarose was purchased from Santa Cruz Biotechnology (Shanghai, China); anti-vascular cell adhesion molecule (VCAM)-1, anti-caspase-1, anti-RelA (phosphor-S536), and anti-vinculin antibodies were purchased from Abcam; anti-IL-1β and anti-IL-18 antibodies were purchased from Sab Biotech (Nanjing, China); anti-NLRP3, anti-RelA and anti-phosphor-IκВα (ser 32) antibodies, and a NF-κВ non-canonical pathway antibody sampler kit including anti-IKKα, anti-RelB and anti-p52 antibodies were purchased from Cell Signaling Technology (Shanghai, China); anti-ASC, anti-IκВα, and anti-p50 antibodies were purchased from Proteintech Group (Wuhan, China). Other reagents, unless otherwise stated, were purchased from Sigma-Aldrich (Shanghai, China).
7
Extraction and Analysis of NF-κB from Small Intestine Tissue
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Small intestine tissues were homogenized in lysis buffer (50 mM Tris and pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM ethylenediaminetetraacetic acid, 1 mM Na3VO4, 0.5-μg/mL leupeptin; Solarbio Science & Technology Co., Ltd. Beijing, China) and centrifuged for 5 min at 12,000 rpm and 4°C. The supernatant was removed and protein in the pellet was quantified using a BCA assay Kit (Solarbio Science & Technology Co., Ltd.). Proteins (30 μg) were resolved using 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Nonspecific reaction of the membrane was removed by blocking it for 1 hr on ice in 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS). NF-κB (Cell Signaling Technology, Waltham, MA, USA) and β-actin (Cell Signaling Technology) were incubated overnight at 4°C in TBS with 3% BSA. Horseradish peroxidase-labeled secondary antibody (Biotime) was diluted 1:5,000 and added. Blots were developed for visualization using an enhanced chemiluminescence detection kit (Thermo Fisher, San Diego, CA, USA). GIS software (Tanon, Shanghai, China) was used to quantify expression.
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