Western blotting was performed as previously described (26 (link)). Briefly, total proteins were extracted from PASMCs and homogenized lung tissue samples using a lysis buffer containing protease (Beyotime Institute of Biotechnology) and phosphatase inhibitors (Cell Signaling Technology, Inc.). Protein concentrations were measured using the BCA method. Proteins (50 µg) were separated via 10% SDS-PAGE and electrophoretically transferred to equilibrated PVDF membranes using semi-dry transfer. Following blocking with 5% skimmed milk for 3 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies targeted against: GAPDH, Notch-3, Jagged-1, Hes-5 and PCNA (Table SII ). Subsequently, the membranes were incubated with anti-rabbit IgG HRP-conjugated antibody (1:200, cat. no. 31470; Invitrogen; Thermo Fisher Scientific, Inc.) or anti-mouse IgG HRP-conjugated antibody (1:800, cat. no. A-11077; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit (cat. no. 32132X3; Invitrogen; Thermo Fisher Scientific, Inc.). GAPDH was used as the loading control. Detection was performed using the LI-COR Odyssey Scanning Infrared Fluorescence Imaging system (LI-COR Bioscience).
Phosphatase inhibitor
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphatase inhibitors are chemical compounds that block the activity of phosphatase enzymes. Phosphatases play a vital role in various cellular processes by removing phosphate groups from proteins, thereby regulating their function. The core function of phosphatase inhibitors is to inhibit the activity of these enzymes, which can be useful in research applications where maintaining the phosphorylation status of proteins is important.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Cell Signaling Technology (7 mentions)
Ripa buffer, by Cell Signaling Technology (4 mentions)
Protease inhibitor, by Merck Group (4 mentions)
Bca assay, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Micro bca assay, by Thermo Fisher Scientific (4 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Cell lysis buffer, by Cell Signaling Technology (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Vero e6 cell, by American Type Culture Collection (2 mentions)
Lysis buffer, by Cell Signaling Technology (2 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (2 mentions)
Irdye 680 800cw anti, by LI COR (2 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (2 mentions)
44 protocols using phosphatase inhibitor
1
Western Blotting of Key Signaling Proteins
2
Immunoprecipitation to Detect hSSB1-hOGG1 Interaction
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To detect an association between hSSB1 with hOGG1, immunoprecipitation from U2OS cell lysates were performed as previously described (20 (link)). Protein concentration was determined by bicinchoninic acid (BCA) assay (Sigma). Equal amounts of lysate were incubated with the appropriate antibody or isotype IgG overnight at 4°C. Antibodies were captured using magnetic protein A/G Dynabeads (Invitrogen), washed five times in NP40 buffer (20mM HEPES, pH 8.0, 150 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 0.2% NP40, 5% glycerol) containing protease inhibitor cocktail (Sigma) and phosphatase inhibitors (Cell Signaling Technology) prior to immunoblot analysis.
3
Immunoblotting Assay Protocol for Protein Analysis
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For the immunoblotting assay, the stimulated cells were washed with 1× PBS and lysed in 1× RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 0.6% Na‐deoxycholate, 1% Triton X‐100) containing protease inhibitors (Calbiochem, 535140) and phosphatase inhibitors (Cell Signalling, 5870 S). Lysed samples were incubated on ice for 15 min followed by centrifugation at 12,000× g for 15 min at 4°C. The protein lysates were determined using a Bradford protein assay (Bio‐Rad Laboratories, 5000006). Protein samples (25 ~ 50 μg) were separated by SDS‐PAGE and then transferred to nitrocellulose membranes (GE Healthcare, 10600002). The membranes were blocked with 5% non‐fat milk in TBS with 0.1% Tween‐20 at RT for 1 h, followed by incubation with specific primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary antibodies at RT for 1 h. The membranes were visualized with Pierce ECL kits (Themo Fisher, 34580) and collected by chemiluminescence imaging system (Uvitech System). The primary and secondary antibodies used for the immunoblotting assay are described in Table S2 .
4
Protein Signaling Pathway Analysis
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Cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA) lysis buffer supplemented with phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor (Cell Signaling), and phosphatase inhibitors (Sigma). Lysates were separated by SDS-PAGE (BioRad, Hercules, CA, USA), transferred to nitrocellulose or polyvinylidene fluoride (PVDF) (BioRad) membranes, and probed with the following primary antibodies: phospho-TrkA(Y674/675), phospho-TrkA(Y785), TrkA, phospho-MEK, MEK, phospho-ERK, ERK, phospho-AKT, AKT, phospho-GSK3b, phospho-Jak1, Jak1, phospho-Stat3, Stat3 (Cell Signaling, Danvers, MA, USA), phospho-TrkC, total TrkC (Thermo Fisher), PD-L1 (Abcam, Cambridge, MA, USA), and actin (ProteinTech, Rosemont, IL, USA).
5
Western Blot Analysis of Signaling Proteins
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Protein lysate was prepared using a Triton X-100 based cell lysis buffer containing proteinase inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Cell Signaling Technology). For protein separation, a standard molecular weight marker and an equal amount of protein from each sample were loaded on a NuPAGE Novex 4–12% Bis-Tris SDS–PAGE gel (Thermo Fisher Scientific Inc.), followed by wet transfer onto a 0.45 µm nitrocellulose membrane which was subsequently blocked with a 5% BSA in PBST solution for 30 min. Overnight incubation with a specific primary antibody at 4 °C was followed by incubation with a secondary antibody conjugated with IRDye 680RD or IRDye 800CW (LI-COR Biosciences) for 1 h at RT. Target proteins were detected using the Odyssey CLx Imaging System (LI-COR Biosciences). β-actin was used as a control for all western blots. Primary antibodies used: β-actin (1:5000, Cat. No. 3700), Akt (1:1000, Cat. No. 9272), phospho-Akt (1:1000, Cat. No. 4051), Erk (1:1000, Cat. No. 4695), phospho-Erk (1:1000, Cat. No. 9101), phospho-FRS2α (1:1000, Cat. No. 3864) from Cell Signaling Technology, and FGF10 (1:2000, Cat. No. ABN44) from Merck Millipore.
6
Western Blot Analysis of IL6
7
Western Blot Analysis of Cellular Proteins
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Mesenchymal stem cells or RMA-mOVA cells were lysed directly on the 10 cm petri dishes or in pellet by M-PER™ mammalian protein extraction reagent (#78501, Thermo Fischer Scientific) containing cocktails of protease (#04693159001, Roche Life Science) and phosphatase inhibitors (#5872, Cell Signaling Technology) after being washed by 1× PBS for two times. Cell debris was removed by 13000 rpm. Centrifugation was done for 10 min. Supernatants containing proteins of interest were analyzed by Western blotting by using mini-gel system from BioRad for the SDS-PAGE. Acrylamide gels were prepared in 10% of acrylamide concentration. Proteins were transferred by Trans-Blot® SD Semi-Dry Transfer Cell and detected by specific primary antibodies and HRP-conjugated antibodies. The results were visualized by HRP substrates (#WBKLS0100, Merck Millipore) and optical films according to the manufacturer’s guides.
8
Protein Expression Analysis in Vascular Cells
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Proteins were extracted from the isolated mesenteric vessel beds of mice or from the cultured human coronary artery SMCs (Lonza, Walkersville, MD) by using RIPA buffer (Cell Signaling Technology, Inc., Boston, MA) supplemented with complete protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitors (Cell Signaling Technology, Inc., Boston, MA). Western blotting were performed as we described previously [15 (link)] by using antibodies to Src, phospho-Src (Tyr416), MLC, and phospho-MLC (Ser19) (Cell Signaling Technology, Inc., Boston, MA). Western blotting bands were scanned with HP Scanjet 7400c, and the band intensities were quantified with ImageJ software.
9
Western Blot Analysis of NLRP3 Inflammasome
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Culture supernatants of DCs treated with MTX and TCL for 2 h were collected and precipitated with equal volumes of methanol and 1/4th volume of chloroform. The solutions were vortexed and centrifuged for 15 min at 20,000 × g, and the upper phase was discarded. After adding 600 μL methanol to the interphase, the mixture was centrifuged for 15 min at 20,000 × g, and the resulting protein pellets were dried at 55°C and re-suspended in loading buffer. The cell pellets were homogenised with a cell lysis buffer containing Phenylmethylsulfonyl fluoride (PMSF), complete protease inhibitors and phosphatase inhibitors (Cell Signaling Technology). The lysates were quantified by BCA assay (Thermo Fisher Scientific), and equal amounts of proteins were mixed with the loading buffer. All protein samples were boiled for 5 min, separated using a 12 % SDS-PAGE gel and then transferred onto nitrocellulose membranes. The blots were incubated with primary antibodies against caspase-1, cleaved caspase-1, IL-1β, cleaved IL-1β, NLRP3, and β-actin at the recommended dilutions, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The bands were visualised using enhanced chemiluminescence reagents from Cell Signaling Technology and Abcam.
10
Western Blot Analysis of Protein Signaling
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INS-1-3 cells cultured in different conditions were lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche, USA) and phosphatase inhibitors (Cell Signaling Technology, USA). Total protein concentrations were determined by a BCA protein assay kit (Beyotime, China). Aliquots of 30 μg proteins were loaded on 10% SDS-polyacrylamide electrophoresis and then transferred onto PVDF membranes. Primary antibodies used were anti-p38 (1 : 1000; CST, USA), anti-phospho-p38 (1 : 1000; CST, USA), and anti-β-actin (1 : 20000; Sigma, USA). β-actin was used for normalization of protein loading. Free and polymerized tubulin fractions were probed with anti-α-tubulin (1 : 200; Santa Cruz, USA). Band intensities were quantified by densitometry using the NIH program Image J®.
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