Journal: bioRxiv

Article Title: Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC, and metalloprotease-mediated cleavage

doi: 10.1101/2024.04.29.591680

Figure Lengend Snippet: A, Cloning strategy and development of the ecto-TrIP Fc-fusion protein used for immunization and antibody development. B, SDS-PAGE of the ectoTrIP-Ig fusion protein under reducing and non-reducing conditions. C, Flow cytometry data showing the binding of each of 3 anti-TrIP monoclonal antibodies to a Flag-tagged WT murine TrIP construct expressed in HEK293T cells. D, Flow cytometry data showing staining of the 3 clones on HEK293T cells transfected with a plasmid encoding Flag-tagged human TrIP. E, Flow cytometry data showing the staining of each of the 3 anti-TrIP mAbs on HEK293T cells transfected with a Flag-tagged mTrIP construct lacking the extracellular kringle domain (ΔKringle).

Article Snippet: Monclonal antibodies to the ecto domain of murine TrIP were developed in conjunction with a commercial vendor (Rockland, Inc.).

Techniques: Cloning, SDS Page, Flow Cytometry, Binding Assay, Bioprocessing, Construct, Staining, Clone Assay, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC, and metalloprotease-mediated cleavage

doi: 10.1101/2024.04.29.591680

Figure Lengend Snippet: A, WT C57BL/6 splenocytes were stimulated with plate-coated anti-CD3 and anti-CD28 at the indicated concentrations and TrIP loss on the CD8 + T cells was followed over time by flow cytometry. B, Histogram of the TrIP expression on CD8 + T cells at the 4-hr timepoint of each dose. C, WT B6 splenocytes stimulated with 2 μg/ml of plate-coated anti-CD3 in the presence or absence of saturating amounts of CTLA4-Ig (20 μg/ml). D, Representative histogram overlay of CTLA4-Ig blockade experiment at the 1-hr timepoint. E, WT P14 TCR Tg splenocytes were stimulated with 100 ng/ml of the indicated cognate peptide variants (peptide affinity: Hi-Aff gp33>WT gp33>L6F gp33) and followed TrIP expression on the CD8 + T cells over time via flow cytometry. F, Representative TrIP staining of each peptide stimulation at the 4-hr timepoint, shown by both dot plot and histogram overlays. G, WT P14 splenocytes stimulated with 200 ng/ml of WT gp33 peptide showing staining for pS6(S235/S236) vs. TrIP staining in CD8 + T cells following activation. H, Quantification of pS6(S235/S236) MFI during the 4 hours of stimulation. I, CD69 vs TrIP expression from the same stimulation described in G-H. J, Frequencies of total TrIP + and total CD69 + cells. Ordinary two-way ANOVA used for statistical comparisons (p: *<0.05, **<0.01, ***<0.001, ****<0.0001).

Article Snippet: Monclonal antibodies to the ecto domain of murine TrIP were developed in conjunction with a commercial vendor (Rockland, Inc.).

Techniques: Flow Cytometry, Expressing, Staining, Activation Assay

Journal: bioRxiv

Article Title: Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC, and metalloprotease-mediated cleavage

doi: 10.1101/2024.04.29.591680

Figure Lengend Snippet: D10 T cells were co-transfected with pMaxGFP alone or together with the indicated TrIP plasmid constucts. Twenty four hours following transfection, cells were stimulated with 10 μg/ml plate-coated anti-CD3 + anti-CD28 or PMA/ionomycin (50 ng/mL and 500 ng/mL, respectively). A, AlphaFold-computed predicted crystal structure of TrIP protein, indicated is the model confidence by color. B, Diagram depecting the two truncation mutants used in these studies. All flow plots show cells gated on GFP + , C-F, D10 T cells were transfected with GFP control alone (C) or together with FL TrIP (D) or the 16aa (E) or 29aa (F) deletions of TrIP. The next day, cells were stimulated as indicated at the top of each column, for one hour, and cells were stained with antibodies to Flag and pS6 (S235/S236). Contour plots are based on gating of GFP+ cells. G, Quantification of ther percentage of Flag+ cells in the transfected D10 cells following αCD3/CD28 stimulation. H, Quantification of pS6(235/236) MFI in transfected cells after αCD3/CD28 stimulation.

Article Snippet: Monclonal antibodies to the ecto domain of murine TrIP were developed in conjunction with a commercial vendor (Rockland, Inc.).

Techniques: Transfection, Plasmid Preparation, Control, Staining