Journal: Cell reports methods
Article Title: Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation.
doi: 10.1016/j.crmeth.2023.100518
Figure Lengend Snippet: Figure 4. Effect of hexosamine biosynthetic pathway hyperactivity on cellular UDP-GlcNAc and protein O-GlcNAcylation in AML12 cells (A–C) UDP-GlcNAc content (A), protein O-GlcNAcylation (B), and OGA-to-OGT expression ratio (C) in parental cells and cells with stable overexpression of wild- type (WT) or E328K mutant GFPT1. (D) Representative western blots and total protein staining as a loading control. One-way ANOVA followed by the selected pairwise comparisons (t test). Data points represent replicate cell culture flasks. Error bars represent mean and ±SD.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Clone RL2 Biolegend Cat#677902; RRID:AB_2565912 Clone CTD110.6 Biolegend Cat#838004; RRID:AB_2629520 GFPT1 Abcam Cat#ab125069; clone EPR4854; RRID:AB_10975709 OGT Abcam Cat#ab96718; RRID:AB_10680015 OGA Merck Cat#HPA036141: RRID:AB_10672079 a-tubulin Cell Signaling Technology Cat#3873; clone DM1A; RRID:AB_1904178 Anti-mouse IgG peroxidase conjugate Cell Signaling Technology Cat#7076; RRID:AB_330924 Anti-mouse IgM peroxidase conjugate Jackson Immunoresearch Cat#115-035-020; RRID:AB_2338502 Anti-rabbit IgG peroxidase conjugate Cell Signaling Technology Cat#7074; RRID:AB_2099233 Bacterial and virus strains E. coli BL21 in-lab N/A E. coli DH5alpha in-lab N/A Chemicals, peptides, and recombinant proteins Human Recombinant OGT R&D systems Cat#8446-GT Alkaline phosphatase Thermo Scientific Cat#EF0651 Antarctic phosphatase New England BioLabs Cat#M0289 Human casein kinase II New England BioLabs Cat#P6010 Benzonase Sigma-Aldrich Cat#E1014 cOmpleteTM, EDTA-free Protease Inhibitor Cocktail Roche Cat#04693132001 HisPur Cobalt resin Thermo Scientific Cat#89964 GlcNAc-acceptor peptide (KKKYPGGSTPVSSANMM) Nordic BioSite Custom synthesis Nunc MaxiSorpTM 384 Sigma-Aldrich Cat#P6116 UDP-GlcNAc Sigma-Aldrich Cat#U4375 Amplex UltraRed Invitrogen Cat#A36006 Experimental models: Cell lines AML12 mouse hepatocyte cell line ATCC Cat#CRL-2254 AML12 cells overexpression wild-type GFPT1 This paper N/A AML12 cells overexpressing E328K mutant GFPT1 This paper N/A TU8988T pancreatic adenocarcinoma cell line Kim et al. 202138 N/A GFPT1 knockout TU8988T cells Kim et al. 202138 N/A HCT 116 Homo human colorectal carcinoma ATCC Cat#CCL-247 NIH/3T3 mouse embryonic fibroblast ATCC Cat#CRL-1658 Hepa1-6 mouse hepatoma ATCC Cat#CRL-1830 293T human kidney epithelium ATCC Cat#CRL-3216 HeLa human adenocarcinoma ATCC Cat#CRM-CCL-2 Experimental models: Organisms/strains Congenic C57BL/6JCrl mice Harlan RRID:IMSR_JAX:000664 Oligonucleotides Human OGT PCR cloning MunI-OGT 50-ATCAATTGACCA TGCTGTGTCCCACCCATGCAGA-30 Metabion N/A (Continued on next page) Cell Reports Methods 3, 100518, July 24, 2023 e1
Techniques: Expressing, Over Expression, Mutagenesis, Western Blot, Staining, Control, Cell Culture
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