On Day 1 of subculture, the medium in the apical chamber was replaced with 100 µL of EC medium lacking bFGF and RA and containing 3 µM of leptin and 3 mM of amino acid substrate. After 30 min, 100 µL of medium was collected from the basolateral chamber and stored at -80 °C. ELISA was then used to quantify leptin concentration as previously described. In terms of amino acids, L-Cystine (Sigma-Aldrich #C8755) was selected as the substrate of cystine/glutamate transporter (encoded by SLC7A11), L-Serine (Sigma-Aldrich #S4311) was selected as the substrate of neutral amino acid transporter A (encoded by SLC1A4) and neutral amino acid transporter B(0) (encoded by SLC1A5), and glycine (Sigma-Aldrich #G8790) was selected as the substrate of glycine transporter 1 (encoded by SLC6A9). CAT-1 (encoded by SLC7A1) and CAT-3 (encoded by SLC7A3) had 4 substrates including L-Lysine hydrochloride (Fisher Scientific #BP386), L-Histidine (Sigma-Aldrich #H6034), L-Ornithine monohydrochloride (Sigma-Aldrich #O6503), and L-Arginine (Acros Organics #104,991,000).
G8790
Manufactured by Merck Group
The G8790 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of the G8790 is to provide a reliable and consistent solution for laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
L arginine, by Thermo Fisher Scientific (1 mentions)
L serine, by Merck Group (1 mentions)
L cystine, by Merck Group (1 mentions)
L histidine, by Merck Group (1 mentions)
L ornithine monohydrochloride, by Merck Group (1 mentions)
L lysine hydrochloride, by Thermo Fisher Scientific (1 mentions)
Truchip chromatin shearing kit, by Covaris (1 mentions)
H3k27ac antibody, by Abcam (1 mentions)
Protein a coated beads, by Thermo Fisher Scientific (1 mentions)
F1635, by Merck Group (1 mentions)
2 protocols using g8790
1
Amino Acid Transport Assay in Cells
2
ChIP-Seq Profiling of H3K27Ac Marks
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A total of 20 million MyPL1 and MyPL2 cells were cross‐linked with 1% formaldehyde (Sigma‐Aldrich, F1635) for 10 min at room temperature, followed by a 5‐min treatment with 0.125 M glycine (Sigma‐Aldrich, G‐8790). Nuclei were isolated and chromatin was purified by chemical lysis. The purified chromatin was fragmented into 200–300 bp fragments by sonication (Covaris, S220) using the truChIP chromatin shearing kit (Covaris). Chromatin immunoprecipitation was performed by incubating the chromatin fraction overnight with 100 μL of protein‐A coated beads (Thermo‐Scientific, 53,139) and 8 μg of the H3K27Ac antibody (Abcam, ab4729). The next day, beads were washed to remove nonspecific binding events and enriched chromatin fragments were eluted from the beads, followed by reverse cross‐linking by incubation at 65°C overnight. DNA was subsequently purified by phenol/chloroform extraction, assisted by phase lock gel tubes (5Prime). DNA obtained from the ChIP assays was adaptor‐ligated, amplified, and analyzed by Illumina sequencing. Raw sequencing data were mapped to the mouse reference genome (GRCm38) using Bowtie. Peak calling was performed using MACS2.
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