The functional role of the most commonly expressed L-AA receptor, CaSR, was tested by measuring the release of GLP-1 from STC-1 cells treated with siRNA for CaSR. STC-1 cells were transfected with control siRNA (SC-37007) or siRNA to CaSR (SC-44374) from Santa Cruz by incubation with siRNA at a concentration of 60 pM for 48 hours. The cells were then incubated in DMEM containing protease and phosphatase inhibitors (Abcam #AB201119) with or without 10 mM L-phenylalanine for 3 hours. The supernatant was collected and GLP-1 was measured by ELISA according to the directions of the manufacturer (GLP-1 EIA kit #RAB0201; Sigma Chemicals, St Louis, MO). The ability of siRNA to suppress the CaSR expression was confirmed in two ways. STC-1 cells were collected from some wells, protein was extracted, and Western blots performed to determine CaSR expression using antibody to CaSR (AB#19347; 1:1000 dilution; Abcam, Cambridge, UK). In other wells, the STC-1 were immunostained with the same CaSR antibody at a dilution of 1:300 as described above.
Ab201119
Manufactured by Abcam
Sourced in United Kingdom
Ab201119 is a lab equipment product offered by Abcam. It is a core tool used for a specific purpose in the laboratory setting. The description of this product is limited to its core function without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab156034, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab19347, by Abcam (1 mentions)
Glp 1 eia kit, by Merck Group (1 mentions)
Sc 37007, by Santa Cruz Biotechnology (1 mentions)
Piercetm bca protein assay, by Thermo Fisher Scientific (1 mentions)
Ipvh00010, by Cytiva (1 mentions)
Ecl chemiluminescence solution, by Biosharp (1 mentions)
M per reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch gel and western blot imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Imaging analyzer, by Bio-Rad (1 mentions)
N cadherin, by Beyotime (1 mentions)
Vimentin, by Beyotime (1 mentions)
Ikβ α, by Proteintech (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Ecl plus kit, by Beyotime (1 mentions)
Bca assay, by Beyotime (1 mentions)
7 protocols using ab201119
1
Functional Role of CaSR in GLP-1 Release
2
Protein Expression Analysis by Western Blot
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After transfected and cultured for 48 h, cells were washed with phosphate-buffered saline (PBS) and lysed by M-PER reagents (78501, Pierce Chemical, IL, USA) containing protein inhibitors (ab201119, Abcam, Cambridge, UK)). Protein quantification was achieved by Pierce TM BCA Protein Assay (UD281372; Thermo Fisher Scientific, Waltham, MA, USA). Samples with loading buffer were loaded onto a 10% SDS-PAGE, then electrotransferred to the PVDF membrane (IPVH00010; Amersham Biosciences, NJ, USA). Then membranes were cut due to molecular weight and incubated with primary antibodies at 4 °C overnight. The membranes were washed with TBST and hybridized with secondary antibodies for one hour at room temperature. Finally, ECL chemiluminescence solution (SW2030, Biosharp, Hefei, China) exposure made the protein band visualized through an automatic chemiluminescence system (5200, Tanon, Shanghai, China). Relative protein expression levels were qualified to GAPDH, and data were obtained from independent triplicate samples. Antibodies used in this study were listed in Supplementary Table S4 .
3
MMP-1 Protein Expression in PdLFs
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To investigate the protein expression of MMP-1 in PdLFs, the cells were treated with 1 µM, 10 µM or 100 µM l -lactic acid compared to 1 µM PMA for 2 and 6 days and the western blot was performed according to Hamoudi et al.75 (link). The cell pellets were lysed in RIPA buffer (ab156034, Abcam, UK) supplemented with 1:10 protease and phosphatase inhibitor cocktail-EDTA free (ab201119, Abcam, UK). The protein lysates were quantified using the Pierce BCA protein assay kit (Thermo-Scientific, USA). 10 µg Protein samples were used for MMP-1 detection using MMP-1 (54,376) rabbit monoclonal antibody and normalized with β-actin (13E5) rabbit monoclonal antibody according to the manufacturer’s instructions. The blots were visualized using the Clarity Western ECL Substrate (Bio-Rad, USA) in the ChemiDoc Touch Gel and Western Blot Imaging System (Bio-Rad, USA). Image Lab Software (V 6.1.0) was used to detect and quantify the protein bands.
4
Western Blot Analysis of Protein Markers
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Total proteins were extracted from both cell types using RIPA lysis buffer containing protease-phosphatase inhibitors (ab201119, abcam, the UK), and PMSF (Solarbio, Beijing, China) and placed on ice for 15 min. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked with 5% skim milk in tris-buffered saline containing 0.2% Tween-20 (TBST) for 40 min and were then treated with monoclonal rabbit antibodies anti TRAF5 (1:400, ProteinTech, Wuhan, China), IKβ-α (1:200, ProteinTech, Wuhan, China), N-cadherin (1:200, Beyotime, Nantong, China), Vimentin (1:200, Beyotime, Nantong, China) and MMP7 (1:200, Beyotime, Nantong, China), and monoclonal mouse GAPDH (1:1,000, ThermoFisher, Carlsbad, USA) and incubated overnight at 4 °C. After washing three times with TBST for 15 min, membranes were incubated with the secondary antibody for 40 min at room temperature. After washing three times with TBST for 10 min, the immunoreactive bands were visualized using ECL Plus kit (Beyotime, Nantong, China) and the imaging analyzer (Bio-rad, USA) was used for observation and image acquisition. GAPDH was used as a loading control.
5
Protein Extraction and Quantification
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Cells were washed 3 times with ice-cold PBS, scraped in cold PBS, collected by centrifugation at 300 × g, then incubated in a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1.0 mM EDTA, 0.1% Triton-X100 [Sigma, X100], protease and phosphatase inhibitors [abcam, ab201119]) for 30 min on the ice. Lysates were sonicated for 5 s 3 times and centrifuged at 13000 × g for 10 min. The supernatant fractions were collected and determined for the protein concentration by using the BCA assay (Beyotime, P0010). The following antibodies for western blot were used: anti-PARK7, anti-ACTB, anti-LAMP2A, anti-HSPA8, and anti-LAMP1.
6
Stepwise Extraction of Hippocampal Proteins
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Hippocampal grey matter from snap-frozen human AD and control samples was dissected by carefully scoring the tissue block with a fine scalpel and subsequent isolation of the tissue by cryosectioning in a Leica cryostat. The resultant tissue samples were homogenized in RIPA buffer (ab156034, Abcam) containing protease and phosphatase inhibitors (ab201119, Abcam) and incubated on ice for 20 min at 4 °C. The protein extract was centrifuged at 14,800 g for 15 min at 4 °C. The resulting supernatant was taken as the RIPA soluble fraction. Pellets were washed in TBS and homogenized in 6 M urea/5% SDS for 30 min at room temperature (RT) and centrifuged at 14,800 g for 10 min at 4 °C. The resultant supernatant was the urea fraction. The samples were stored at − 80 °C until further use. To extract proteins in native condition, tissue was homogenized in TBS containing 0.1% of Triton X-100, incubated for 10 min at RT, followed by centrifugation at 16,000 g for 10 min.
7
Liver Mitochondria Isolation Protocol
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The liver samples were cut into small pieces and placed in 10 vol cold Buffer A (1 mg/mL fatty acid-free BSA, 75 mmol/L sucrose, 225 mmol/L mannitol, 10 mmol/L N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (HEPES), and 0.1 mmol/L ethylene glycol tetraacetic acid (EGTA), pH 7.4), which was supplemented with phosphatase and protease inhibitors (ab201119, Abcam, Boston, Massachusetts, USA). After that, sample tissue was homogenized with a Teflon Dounce homogenizer, followed by differential centrifugation to isolate mitochondria (700 × g for 15 min and then 10,000 × g for 15 min at 4 ℃). The resultant supernatant containing cytoplasm was collected for the analysis related to oxidative damage. The sediment containing mitochondria were subsequently resuspended within Buffer B (10 mmol/L HEPES, 0.1 mmol/L EGTA, and 395 mmol/L sucrose, pH 7.4). Total proteins of cytoplasm and mitochondria solutions were measured by the BCA method using the total protein quantitative assay kit (A045-4–2, Nanjing Jiancheng Institute of Bioengineering, Jiangsu, China).
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