Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet:

Article Snippet: Protein G Sepharose 4 Fast Flow , GE Healthcare , Cat# 17-0618-01.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Modification, Protease Inhibitor, Polymer, Ligation, Labeling, Reverse Transcription, Purification, Plasmid Preparation, Control, Software

Journal: Scientific Reports

Article Title: N- glycosylation of α 1D -adrenergic receptor N-terminal domain is required for correct trafficking, function, and biogenesis

doi: 10.1038/s41598-020-64102-4

Figure Lengend Snippet: Site-directed mutagenesis and lectin affinity purification analyses reveal α 1D -AR is dually glycosylated at N65 and N82. ( A ) Lysate from HEK293 cells expressing WT SNAP-α 1D ( input ) was incubated with lentil lectin sepharose beads to isolate glycosylated proteins. Bound protein was eluted with methyl-α-D-mannopyranoside ( bound ), and analyzed using PAGE NIR. SNAP-α 1D monomers (►) and higher order oligomers, as well as the previously described N-terminal cleavage product, are present in the elutant. ( B ) Schematic and ( C ) PAGE NIR of HEK293 cell lysate expressing WT, single glycosylation mutants (N65Q and N82Q), and double glycosylation mutant (NQQ) SNAP-α 1D species. ( D ) HEK293 cells expressing WT SNAP-α 1D were incubated for 16 hr with vehicle (−) or tunicamycin (TUN, +), and analyzed with PAGE NIR. A signal at 43 kDa was observed in the tunicamycin treated samples (○). ( E ) PAGE NIR of HEK293 cell lysate transfected with N-terminal SNAP-epitope ( λ = 800 nm, green ) and C-terminal CLIP-epitope ( λ = 700 nm, red ) dual tagged WT (S-WT-C) and NQQ (S-NQQ-C) α 1D -AR constructs. ( F,I ) PAGE NIR of HEK293 cell lysate expressing S-WT-C or ( G,J ) S-NQQ-C following 24 hr bortezomib (BTZ) ( F,G ) or protease inhibitor (PI) treatment ( I,J ). ( H ) Quantitation of signals from F and G normalized to vehicle. ( K ) Quantitation of fluorescent signals from I and J normalized to vehicle. All gels are representative images from n = 3 experiments. For F and G, data are represented as mean ± SEM; Unpaired t tests, ***p < 0.001.

Article Snippet: The soluble fraction was incubated with Lentil Lectin Sepharose 4B beads (GE Healthcare, Chicago, IL) and 1 μL of 25 μL BG-782 for 1 hr at room temperature.

Techniques: Mutagenesis, Affinity Purification, Expressing, Incubation, Glycoproteomics, Transfection, Construct, Protease Inhibitor, Quantitation Assay