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: 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: Mouse Gfpt1 PCR cloning: Gfpt1-STP-XhoI 5′-ATCTCGAGTTAC TCTACTGTTACAGATTTGGC-3′ , Metabion , N/A.

Techniques: Expressing, Over Expression, Mutagenesis, Western Blot, Staining, Control, Cell Culture

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: Effect of disrupted hexosamine biosynthetic pathway on cellular UDP-GlcNAc and protein O-GlcNAcylation in a pancreatic adenocarcinoma cell line (TU8988T) (A–C) UDP-GlcNAc content (A), protein O-GlcNAcylation (B), and OGA-to-OGT expression ratio (C) in parental and GFPT1 knockout cells with and without 10 mM GlcNAc in media. Note the logarithmic y axis in (A). The indicated time points refer to duration since replacement of the culture media (start of the GlcNAc starvation). (D) Representative western blots and total protein staining. (E) Growth curves of the GFPT1 knockout cells. ∗Bonferroni-corrected p < 0.0001 (1-way ANOVA followed by the selected pairwise comparisons). The data points represent replicate cell culture flasks. Error bars represent ±SD.

Article Snippet: Mouse Gfpt1 PCR cloning: Gfpt1-STP-XhoI 5′-ATCTCGAGTTAC TCTACTGTTACAGATTTGGC-3′ , Metabion , N/A.

Techniques: Expressing, Knock-Out, Western Blot, Staining, Cell Culture

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:

Article Snippet: Mouse Gfpt1 PCR cloning: Gfpt1-STP-XhoI 5′-ATCTCGAGTTAC TCTACTGTTACAGATTTGGC-3′ , Metabion , N/A.

Techniques: Virus, Recombinant, Protease Inhibitor, Over Expression, Mutagenesis, Knock-Out, PCR Cloning, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Software

Journal: Gene

Article Title: Enrichment of meiotic recombination hotspot sequences by avidin capture technology 2

doi: 10.1016/j.gene.2012.12.042

Figure Lengend Snippet: Principle of the capture protocol. The hybridization of oligos 1 and 2 creates an EcoRI restriction site and a 5’ end overhang that is complementary to the sequence in the recombination hotspot 5’-CCN CCN TNN CCN C-3’. The annealed capture oligonucleotides are immobilized on streptavidin-magnetic coated beads through the biotin-conjugated 5’ end in oligo 1.

Article Snippet: Oligos were denatured by heating at 95°C for 5 min in a thermocycler, followed by annealing at 22°C for 1 h. The annealed oligos have an EcoRI restriction site ( ).

Techniques: Hybridization, Sequencing

Journal: Gene

Article Title: Enrichment of meiotic recombination hotspot sequences by avidin capture technology 2

doi: 10.1016/j.gene.2012.12.042

Figure Lengend Snippet: Enrichment of a DNA containing the hotspot motif from a mixture of short, double-stranded DNA. (A) Synthetic dsDNA oligonucleotides: 100-mer containing the hotspot sequence for recombination; 50-mer not containing the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). (C) As described for “A” but now the 50-mer contains the hotspot sequence. (D) As described for “B.” Abbreviation: M, marker.

Article Snippet: Oligos were denatured by heating at 95°C for 5 min in a thermocycler, followed by annealing at 22°C for 1 h. The annealed oligos have an EcoRI restriction site ( ).

Techniques: Sequencing, Amplification, Marker

Journal: Gene

Article Title: Enrichment of meiotic recombination hotspot sequences by avidin capture technology 2

doi: 10.1016/j.gene.2012.12.042

Figure Lengend Snippet: Capture of long dsDNA with oligo-Blue. (A) EcoRI fragment of pBluescript II sk (+), containing a 13-mer with a degree of degeneration similar to the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). Abbreviation: M, marker.

Article Snippet: Oligos were denatured by heating at 95°C for 5 min in a thermocycler, followed by annealing at 22°C for 1 h. The annealed oligos have an EcoRI restriction site ( ).

Techniques: Sequencing, Amplification, Marker