Journal: Cell Death and Differentiation

Article Title: Endothelial cells-derived SEMA3G suppresses glioblastoma stem cells by inducing c-Myc degradation

doi: 10.1038/s41418-025-01534-3

Figure Lengend Snippet: A The ubiquitination level of c-Myc in GSC07 cells treated with or without ML141 (10 μmol/l) for 24 h. B , C c-Myc level in T98G cells transfected with GFP-Tagged Cdc42 WT , Cdc42 T17N or Cdc42 Q61L . D Schematic diagram depicting the plasmid construction for bimolecular fluorescence complementation assays. E Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between Cdc42 and WWP2 . F HEK-293 cells were transfected with CrN173-tagged Cdc42 T17N or Cdc42 Q61L , and VC155-tagged WWP2 for 48 h. CFP signal that represents Cdc42 binding to WWP2 were determined using confocal. Scale bar, 5 μm. G HEK-293 cells were transfected with Flag-tagged c-Myc, HA-tagged ubiquitin, MYC-tagged WWP2 and GFP-tagged Cdc42 T17N or Cdc42 Q61L for 48 h. The ubiquitin level of c-Myc and the binding of Cdc42 to WWP2 were determined by co-immunoprecipitation. H Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between WWP2 and Cdc42 or c-Myc. I HEK-293 cells were incubated with or without SEMA3G (200 ng/ml) for 48 h after co-transfected with CrN173-tagged Cdc42 WT or VN173-tagged c-Myc and VC155-tagged WWP2 for 12 h. CFP signal that represents Cdc42 binding to WWP2 and Venus signal that represents WWP2 binding to c-Myc were captured using confocal. Scale bar, 5 μm. J HEK-293 cells that were co-transfected with GFP-tagged Cdc42 WT , MYC-tagged WWP2, Flag-tagged c-Myc, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The binding of Cdc42 WT to WWP2 and the ubiquitination level of c-Myc were determined by co-immunoprecipitation. K HEK-293 cells were co-transfected with GFP-tagged Cdc42 Q61L and MYC-tagged WWP2, Flag-tagged c-MYC, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The protein level of c-Myc and ubiquitination level of c-Myc were determined by co-immunoprecipitation. L Schematic diagram illustrating the potential mechanism by which Cdc42 regulates WWP2 binding to c-Myc and promotes c-Myc degradation. All the western blot bands represent one of the three independent experiments.

Article Snippet: USA), or ML141 (MedChemExpress, NJ, USA) according to the experimental design.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Binding Assay, Immunoprecipitation, Incubation, Western Blot

Journal: Archives of biochemistry and biophysics

Article Title: INCORPORATION OF PHOSPHATE INTO GLYCOGEN BY GLYCOGEN SYNTHASE

doi: 10.1016/j.abb.2016.03.020

Figure Lengend Snippet: Effect of treating 32P-labeled glycogen with laforin or glucosidases. A Glycogen was labeled by incubation with 5 μM [β-32P]UDP-glucose (lower panel) or [U-14C]glucose (upper panel) and yeast glycogen synthase (2 μg/ml) for 30 min. Glycogen was precipitated with ethanol, dissolved in buffer and treated with α-glucosidases (α-amylase and amyloglucosidase) (α-G), inactive mutant laforin (C266S Laf) or wild type laforin (WT Laf) as indicated, and analyzed by SDS-Page (see Materials and Methods). Dried gels were analyzed by a Phosphorimager. C, control reaction lacking glycogen synthase; NT, not treated. B UDP-glucose and UDP were incubated with active (WT) or inactive (C266S) laforin as indicated and analyzed by HPAEC. Chromatograms of UDP, UMP and UDP-glucose standards are shown in the lowermost panel.

Article Snippet: High purity UDP (99.2%) used in laforin reactions was from Chem-Impex International, Inc. (#00310).

Techniques: Labeling, Incubation, Mutagenesis, SDS Page

Journal: Archives of biochemistry and biophysics

Article Title: INCORPORATION OF PHOSPHATE INTO GLYCOGEN BY GLYCOGEN SYNTHASE

doi: 10.1016/j.abb.2016.03.020

Figure Lengend Snippet: Release of 32Pi from 32P-glycogen by laforin. Glycogen was labeled by incubation with 5 μM [β-32P]UDP-glucose (A) or [U-14C]glucose (B) and yeast glycogen synthase (5 μg/ml) for 30 min. Glycogen was precipitated three times with ethanol, treated with PiBind™ resin, purified by gel filtration and dissolved in buffer. The glycogen was incubated for 2 hr with 50 μg/ml laforin (WT Laf) or laforin inactivated by boiling for 5 min (HI Laf). A control (C) lacked laforin. The reaction mixtures were analyzed by TLC using PEI-cellulose plates. Standards of glucose-1-P (14C-G1P), glucose (14C-Glu) and inorganic phosphate (32Pi), labeled with the indicated isotope, were also analyzed.

Article Snippet: High purity UDP (99.2%) used in laforin reactions was from Chem-Impex International, Inc. (#00310).

Techniques: Labeling, Incubation, Purification, Filtration