Journal: EMBO Reports

Article Title: m 6 A modification of mutant huntingtin RNA promotes the biogenesis of pathogenic huntingtin transcripts

doi: 10.1038/s44319-024-00283-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The qPCR was performed on 96-well plates in a final volume of 12 μL using the Premix Ex Taq Probe-based qPCR assay (Takara Biotechnology, ref. RR390A).

Techniques: Knock-In, Mouse Assay, Recombinant, Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction, Purification, RNA Sequencing Assay, Rapid Amplification of cDNA Ends, Hot Start PCR, Staining, Gel Extraction, Methylation, Viability Assay, CyQUANT Assay, Proliferation Assay, Software, Spectrophotometry, Microscopy, Imaging

Journal: Cancer research

Article Title: Targeting glycosylated PD-1 induces potent anti-tumor immunity

doi: 10.1158/0008-5472.CAN-19-3133

Figure Lengend Snippet: (A) Immunoblot of PD-1 expression in human TNBC tumor tissues. Black circle, glycosylated PD-1; arrowhead, non-glycosylated PD-1. (B) Immunoblot of PD-1 expression in shCTRL, shPD-1, and PD-1-re-expressing Jurkat T cells stimulated by PHA overnight. (C) Glycoprotein staining and Coomassie blue staining of PNGase F-treated purified PD-1. Horseradish peroxidase (HRP) and soybean trypsin inhibitor (STI) served as positive and negative control, respectively. (D) Immunoblot of PD-1 in PD-1-expressing Jurkat (Jurkat-PD-1) T cells treated with inhibitors blocking N-linked or O-linked glycosylation as indicated. (E) Schematic diagram of PD-1 amino acid sequence alignment among different species. The four putative NXT motifs are shown in red. (F) Schematic diagram of full-length PD-1. ECD, extracellular domain; ICD, intracellular domain; SP, signal peptide; TM, transmembrane domain. Four putative NXT motifs in the ECD domain are labeled in red. The numbers indicate the amino acid positions. (G) Immunoblot of the protein expression pattern of PD-1 WT and indicated NQ mutants overexpressed in Jurkat T cells.

Article Snippet: To measure PD-1/PD-L1 interaction, recombinant His-tagged PD-1 was incubated with or without Rapid PNGase F (New England BioLabs, Ipswich, MA, USA) in non-reducing buffer for 30 min at 50 °C and then coated on a nickel-coated 96-well plate.

Techniques: Western Blot, Expressing, Staining, Purification, Negative Control, Blocking Assay, Sequencing, Labeling

Journal: Cancer research

Article Title: Targeting glycosylated PD-1 induces potent anti-tumor immunity

doi: 10.1158/0008-5472.CAN-19-3133

Figure Lengend Snippet: (A) In vitro plate-based binding analysis of purified PD-1 with PD-L1 Fc in the absence or presence of PNGase F. (B) Analysis of PD-1/PD-L1 binding by immunoprecipitation. The lysates of 293T cells overexpressing FLAG-tagged PD-1 WT or 4NQ mutant were incubated with PD-L1 Fc and anti-FLAG M2 agarose, and then subjected to IP-Western. (C) Time-lapse microscopy of the dynamic interaction between green fluorescent-labeled PD-L1-Fc and PD-1 WT or 4NQ expressed in 293T cells. (D) Time-lapse evaluation and quantitation of the dynamic interaction between green fluorescent-labeled PD-L1-Fc and PD-1 WT or the indicated mutants expressed in Jurkat cells. (E) Mapping of the glycosylated sites critical for binding with PD-L1 by immunoprecipitation. The lysates of Jurkat cells overexpressing PD-1 WT or the indicated NQ mutants were incubated with pan mouse IgG dynabeads and Fc (mouse IgG) fused human PD-L1, and then subjected to IP-Western. The band intensity was quantified and normalized to show PD-L1 binding levels (right panel). (F) Flow cytometric analysis of the binding of PD-L1 Fc with cell surface PD-1 by using Jurkat T cells expressing PD-1 WT or the indicated NQ mutants. Median Fluorescence Intensity was quantified and normalized to show PD-L1 binding levels (right panel). All data represent mean ± SD from at least three independent experiments. *, P < 0.05.

Article Snippet: To measure PD-1/PD-L1 interaction, recombinant His-tagged PD-1 was incubated with or without Rapid PNGase F (New England BioLabs, Ipswich, MA, USA) in non-reducing buffer for 30 min at 50 °C and then coated on a nickel-coated 96-well plate.

Techniques: In Vitro, Binding Assay, Purification, Immunoprecipitation, Mutagenesis, Incubation, Western Blot, Time-lapse Microscopy, Labeling, Quantitation Assay, Expressing, Fluorescence

Journal: Cancer research

Article Title: Targeting glycosylated PD-1 induces potent anti-tumor immunity

doi: 10.1158/0008-5472.CAN-19-3133

Figure Lengend Snippet: (A) Dot blot analysis of PD-1 antibodies using purified PD-1 treated with or without PNGase F. (B) Schematic diagram of the live-cell imaging PD-1/PD-L1 binding assay in the presence of gPD-1 antibody. (C) Measurement of the neutralizing activity of gPD-1 antibodies using an IncuCyte system. 293T-PD-1 cells were incubated with fluorescent-labeled PD-L1-Fc fusion protein in the presence of gPD-1 antibodies. The binding of PD-1 and PD-L1 was quantified by counting fluorescent objects per mm2 using the Incucyte™ Zoom system every 2 hours. (D) EC50 calculation of STM418 by GraphPad Prism software. Experiments were conducted as described in (C) in the presence of indicated doses of STM418. (E) KD determination of STM418 or nivolumab. (F) Glyco-specificity evaluation of STM418 by immunoblot analysis of PD-1 WT or the indicated NQ mutants over-expressed in 293T cells. (G) Time-lapse evaluation and quantitation of the dynamic interaction between green fluorescent-labeled PD-L1-Fc and PD-1 WT or the indicated mutants expressed in Jurkat cells in the absence of presence of STM418. PD-L1 binding inhibition is represented by the fluorescence intensity ratio of the STM418 to IgG treatment in each cell line. (H) Epitope mapping of STM418 by high-mass MALDI mass spectrometry (CovalX). Sequences on the PD-1 protein shown in red represent the STM418 antibody binding sites.

Article Snippet: To measure PD-1/PD-L1 interaction, recombinant His-tagged PD-1 was incubated with or without Rapid PNGase F (New England BioLabs, Ipswich, MA, USA) in non-reducing buffer for 30 min at 50 °C and then coated on a nickel-coated 96-well plate.

Techniques: Dot Blot, Purification, Live Cell Imaging, Binding Assay, Activity Assay, Incubation, Labeling, Software, Western Blot, Quantitation Assay, Inhibition, Fluorescence, Mass Spectrometry