Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2024.107930

Figure Lengend Snippet: Development and validation of novel monoclonal antibodies to the ecto domain of TrIP. 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 nonreducing conditions. C , flow cytometry data showing the binding of each anti-TrIP monoclonal antibody to a Flag-tagged WT murine TrIP construct expressed in HEK293T cells. D , flow cytometry data showing staining of the clones on HEK293T cells transfected with a plasmid encoding Flag-tagged human TrIP. E , flow cytometry data showing the staining of each of the anti-TrIP mAbs on HEK293T cells transfected with a Flag-tagged mTrIP construct lacking the extracellular kringle domain (ΔKringle). HEK, human embryonic kidney; mAb, monoclonal antibody; mTrIP, murine TrIP; TrIP, transmembrane inhibitor of PI3K.

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

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

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2024.107930

Figure Lengend Snippet: Stimulation strength-dependent downregulation of TrIP on CD8 + T cells. 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-h time point of each stimulation 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 CTLA4ig blockade experiment at the 1-h time point. 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-h time point, 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) versus TrIP staining in the CD8 + T cells following activation. H , quantification of pS6 (S235/S236) MFI during the 4-h course of stimulation. I , in the same WT P14 stimulation described in G and H , flow plots depicting CD69 versus TrIP expression through 6 h of stimulation. 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). MFI, mean fluorescence intensity; pS6, phosphorylation of the S6 ribosomal subunit; TCR, T-cell receptor; TrIP, transmembrane inhibitor of PI3K.

Article Snippet: Monoclonal 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, Fluorescence, Phospho-proteomics

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2024.107930

Figure Lengend Snippet: CRISPR-mediated KO in naïve CD8 T cells confirms a role for ADAM10 in TrIP downregulation and suggests that PKCθ is dispensable. Naïve T cells were transfected with Cas9 ribonucleoprotein’s targeting ADAM10 or PKCθ, then maintained in rIL-7 for 7 days in vitro , followed by stimulation with α-CD3 mAb. A and B , KO efficiency of ADAM10, shown by flow cytometry ( A ) and histogram ( B ), which indicate an increase in basal TrIP expression before stimulation. TrIP expression was assessed on day 7 after nucleofection, prior to stimulation. C and D , expression of TrIP and ADAM10 after stimulation of the indicated cells, at the indicated time points. Samples were gated on total CD8 + cells. E and F , expression of PKCθ and TrIP at day 7 after nucleofection, and before stimulation, shown by flow cytometry ( E ) and histogram ( F ). MFI data in panel F are from the PKCθ + or PKCθ - gate in panel G for WT and KO cells, respectively. G and H , expression of TrIP and PKCθ after stimulation of the indicated cells, at the indicated time points. MFI data in panel H are from the PKCθ + or PKCθ - gate in panel G for WT and KO cells, respectively. I and J , the percentage of TrIP + cells at the indicated time points after stimulation. Cells in panel I (ADAM10 KO) were gated on total CD8 + cells; cells in panel J were gated on PKCθ + or PKCθ - cells in WT and PKCθ KO conditions, respectively. Ordinary two-way ANOVA was used for statistical comparisons ( p : ∗<0.05, ∗∗<0.01, ∗∗∗<0.001, ∗∗∗∗<0.0001). mAb, monoclonal antibody; MFI, mean fluorescence intensity; PKC, protein kinase C; TrIP, transmembrane inhibitor of PI3K.

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

Techniques: CRISPR, Transfection, In Vitro, Flow Cytometry, Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2024.107930

Figure Lengend Snippet: Truncation of the extracellular stalk renders TrIP resistant to cleavage and dampens PI3K signaling. D10 T cells were cotransfected with pMaxGFP alone or together with the indicated TrIP plasmid constructs. 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 sourced predicted crystal structure of TrIP protein, indicated is the model confidence by color. B , diagram depicting the two truncation mutants used in these studies. All flow plots show cells gated on GFP + . C , control GFP-only transfected D10 cells (GFP only) depicted without stim, following 1-h of stimulation with αCD3/CD28 or PMA/ionomycin. D , Co-GFP+WT TrIP (FL TrIP) transfected D10 T cells depicted without stimulation, or following 1-h ofstimulation with αCD3/CD28 or PMA/ionomycin. E , D10 T cells cotransfected with GFP and the Δ16aa mutant of TrIP (16aa del’n) depicted without stim, or following 1-h of stimulation with αCD3/CD28 or PMA/ionomycin. F , D10 T cells cotransfected with GFP + the Δ29aa mutant of TrIP (29aa del’n) depicted without stimulation or following 1-h of stimulation with αCD3/CD28 or PMA/ionomycin. G , quantification of TrIP-Flag (%Flag + ) in transfected D10 cells following αCD3/CD28 stimulation. H , quantification of pS6 (235/236) MFI in the transfected D10 cells following αCD3/CD28 stimulation. MFI, mean fluorescence intensity; PMA, phorbol 12-myristate 13-acetate; pS6, phosphorylation of the S6 ribosomal subunit; TrIP, transmembrane inhibitor of PI3K.

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

Techniques: Plasmid Preparation, Construct, Transfection, Control, Mutagenesis, Fluorescence, Phospho-proteomics

Journal: Journal of the Formosan Medical Association = Taiwan yi zhi

Article Title: Randomized controlled clinical effectiveness of adjunct 660-nm light-emitting diode irradiation during non-surgical periodontal therapy.

doi: 10.1016/j.jfma.2019.01.010

Figure Lengend Snippet: Figure 3 Change in GCF Biomarkers. (A) Absolute IL-1b levels. (B) Absolute MMP-8 levels. (C) Normalized IL-1b levels. (D) Normalized MMP-8 levels. In (CeD), the biomarker levels in T1 and T2 were normalized based on the baseline biomarker level (T0) of the same examined site. (Significant difference compared with T0: #p < 0.05, ##p < 0.01, ###p < 0.001).

Article Snippet: Please cite this article as: Chen Y-W et al., Randomized controlled c diation during non-surgical periodontal therapy, Journal of t j.jfma.2019.01.010 Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to determine IL-1b and MMP-8 levels from each GCF sample following the manufacturer’s instructions (Boster Biological Tech., Pleasanton CA, USA).

Techniques: Biomarker Discovery