Journal: The FEBS journal

Article Title: Pharmacological inhibition and knockdown of O-GlcNAcase reduces cellular internalization of α-synuclein preformed fibrils.

doi: 10.1111/febs.15349

Figure Lengend Snippet: Fig. 5. Effect of OGA knockdown on a-syn PFF uptake. (A) Immunoblot analysis and quantification of OGA protein levels in SK-N-SH cell lysates after either treatment with Thiamet-G or knocking down OGA using specific siRNA oligo duplex as compared to cells treated with a nontarget siRNA oligo duplex as control (n = 3). (B) Immunoblot analyses of global O-GlcNAc levels and quantification showing the efficacy of OGA inhibition with Thiamet-G and its knockdown using siRNA as compared to cells treated with a nontarget siRNA oligo duplex as control (n = 3). (C) Quantification of high-content cellular analysis of the effects of targeted siRNA knockdown of OGA on uptake of a-syn PFFs (6-h uptake using a a-syn PFF concentration that corresponds to 100 nM monomer concentration before fibrillation) as compared to a nontarget siRNA oligo duplex as control. (D, E) High-content cellular analysis images representing the results shown in panel ‘C’ (n = 3). In each single replicate, 12 different automatically preassigned nonoverlapping fields in each well (eight wells for each condition) were imaged. Images from these fields from each condition were selected as representative images. DAPI channel (nucleus) and Cy5 channel (a-syn PFFs) are represented in blue and red. The scale bar represents 100 lm. (**P ≤0.01, ***P ≤0.001, and ****P ≤0.0001). The results in panels ‘A–E’ represent three independent biological repeats (n = 3). Error bars represent mean SD. Student’s t-test was used for statistical comparisons between control (no Thiamet-G treatment or NT-siRNA) and treated conditions (Thiamet-G or OGA siRNA), respectively.

Article Snippet: OGA siRNA oligo duplex (SR307308C: rCGrArGrCrArArArUrArGrUrArGrUrGrUrUrGrUrCrArGTG) and universal scrambled negative control (nontarget, NT) siRNA oligo duplex, as well as siRNA transfection reagent (siTran), were purchased from OriGene (OriGene Technologies, Rockville, MD, USA).

Techniques: Knockdown, Western Blot, Control, Inhibition, Concentration Assay

Journal: Cell chemical biology

Article Title: Detecting and Imaging O-GlcNAc Sites Using Glycosyltransferases: A Systematic Approach to Study O-GlcNAc.

doi: 10.1016/j.chembiol.2018.07.007

Figure Lengend Snippet: Figure 4. Probing Open and Closed O-GlcNAc Sites on Various Recombinant Proteins Using OGT and B3GALNT2 Each protein was loaded into three consecutive lanes, indicated as lane a, b, and c. Lane a contained the recombinant protein only. Lane b contained additional OGT and UDP-GalNAz for detecting open sites. Lane c contained additional OGT, UDP-GlcNAc, B3GALNT2, and UDP-GalNAz for detecting closed sites. AKT1 E. coli was expressed in E. coli. AKT1 Baculo was ex- pressed in Baculovirus. All other proteins were expressed in E. coli. In the lanes indicated with both AKT3 and CK2, samples of AKT3 were loaded first and then chased with samples of CK2 5 min later. All samples were subjected to click chemistry reaction with Click-iT DIBO Alkyne and separated on 4%–20% SDS gradient gels and visualized with TCE under UV (upper panels). The gels were then blotted to nitrocellulose membrane and detected with SA-HRP (lower panels). Recombinant CEBPB is a truncated protein and lacks trypto- phan residues, therefore it is not visible by TCE staining but was detected by OGT and B3GALNT2 staining when probed with SA-HRP. OGT showed some background staining possibly due to self-labeling. By comparing the signal intensities for lanes a, b, and c, it was concluded that AKT1 (both E. coli- and Baculovirus-expressed versions), CK2, CEBPB, and PFKFB3 contained open sites for O-GlcNAcylation.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli/BL21(DE3) ThermoFisher C6000-03 Chemicals, Peptides, and Recombinant Proteins UDP-GlcNAz (UDP-azido-GlcNAc) R&D Systems Cat#ES104 UDP-GalNAz (UDP-azido-GalNAc) R&D Systems Cat#ES103 Biotin alkyne adduct R&D Systems Cat#ES100 Streptavidin conjugated horseradish peroxidase (strep-HRP) R&D Systems 4800-30-06 UDP-GlcNAc Sigma Aldrich Cat#U4375 Streptavidin, Alexa FluorTM 555 ThermoFisher Scientific Cat#S32355 Click-iT DIBO Alkyne ThermoFisher Scientific Cat#C10412 VisULite MAX ECL Western Blotting Substrate R&D Systems Cat#VL002-200 4’,6-diamidino-2-phenylindole (DAPI) R&D Systems 5748 Benzyl-b-GlcNAc Santa Cruz Biotechnology Cat#sc-221296 OGT substrate peptide AnaSpec Cat#AS-63726 Recombinant human B3GALNT2 R&D Systems Cat#1848-GT Recombinant human Casein Kinase 2a (CK2) R&D Systems Cat#7957-CK Recombinant human O-GlcNAc transferase (OGT) R&D Systems Cat#8446-GT Recombinant B. thetaiotaomicron O-GlcNAcase (OGA) R&D Systems Cat6779-GH Recombinant human AKT1 R&D Systems Lot Code: IPC Recombinant human AKT1 R&D Systems Cat#1775-KS Recombinant human AKT2 R&D Systems Lot Code: IQR Recombinant human AKT3 R&D Systems Lot Code: PIB Recombinant human CEBP b R&D Systems Lot Code: OYK Recombinant human PFKFB3 R&D Systems Cat#8566-BP Recombinant human G6PD R&D Systems Lot Code: DGGC Recombinant human GCK R&D Systems Cat#7840-GK Recombinant human FXR R&D Systems Lot Code: OGV Critical Commercial Assays Glycosyltransferase Activity Kit R&D Systems Cat#EA001 Experimental Models: Cell Lines

Techniques: Recombinant, Membrane, Staining, Labeling

Journal: iScience

Article Title: GFAT2 mediates cardiac hypertrophy through HBP-O-GlcNAcylation-Akt pathway

doi: 10.1016/j.isci.2021.103517

Figure Lengend Snippet: Key resources table

Article Snippet: Rabbit polyclonal anti-OGA , Novus Biologicals , Cat#NBP1-81244; RRID: AB_11053819.

Techniques: Plasmid Preparation, Virus, Recombinant, Sequencing, Software, Microscopy