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

Journal: Orthodontics & craniofacial research

Article Title: Extracellular vesicle identification in tooth movement models

doi: 10.1111/ocr.12287

Figure Lengend Snippet: A) Schematic of NanoView Biosciences detection system. Antibodies to EV surface antigens are attached to a signal enhancing surface. EVs are then incubated on the spots and unbound sample is washed free. Bound EVs can then be detected by interferometrics or by fluorescent labeling. B) GCF was collected from the buccal side of the left upper central incisor by a single Periopaper dip following the method described in detail by our group previously (2) and following a procedure approved by the University of Florida Internal Review Board (IRB201600476). The GCF was analyzed using the ExoView Platform on an anti-CD63 spot as described previously (25). GCF was eluted with 100 μl PBS. Twenty μl of the sample was diluted 1:1 with PBS then introduced into the ExoView apparatus. Samples were incubated for 12 H, washed and labeled for 2 H with antibody, washed again and nanoparticles were detected by either light scatter or fluorescence. Round circles indicate particles detected by light scatter. Bright particles were those labeled with anti-CD63. Most of the anti-CD63 labeled EVs were not detected by the interferometric method indicating the CD63 labeled particles are mostly smaller than about 60 nm in diameter, the detection limit of the interferometric technique.

Article Snippet: In preliminary runs of GCF samples (courtesy of NanoView Biosciences, Boston MA) in an ExoView platform, both CD9 and CD63 containing EVs were detected in GCF ( ).

Techniques: Incubation, Labeling, Fluorescence