Journal: Acta Neuropathologica Communications

Article Title: Microglia depletion diminishes key elements of the leukotriene pathway in the brain of Alzheimer’s Disease mice

doi: 10.1186/s40478-020-00989-4

Figure Lengend Snippet: Tested 5-Lox and FLAP antibodies on mouse brain tissue and their immunoreactivity in neurons and/or microglia cells. aa = amino acid; − = no, + = mild, ++ = moderate and +++ = strong immunoreactivity

Article Snippet: 5-Lox (red, using polyclonal 5-Lox antibody from Abcam #ab39347) is expressed in FLAP (green, polyclonal FLAP antibody from Novus Biologicals NB300–891) positive cells associated with the amyloid plaque.

Techniques: Recombinant

Journal: Acta Neuropathologica Communications

Article Title: Microglia depletion diminishes key elements of the leukotriene pathway in the brain of Alzheimer’s Disease mice

doi: 10.1186/s40478-020-00989-4

Figure Lengend Snippet: Immunohistochemical analysis for 5-Lox and FLAP expression in WT and APP-PS1 mouse brains. a In mouse brains the majority of 5-Lox (green, monoclonal 5-Lox antibody from Abcam #ab169755) immunoreactivity was observed in the granular layer of the dentate gyrus and co-localized with NeuN (white). b 5-Lox immunoreactivity (green, using polyclonal 5-Lox antibody from Abcam #ab39347) was also observed outside the granular layer in particular in APP-PS1 mice and co-localized with some Iba1 positive microglia (white) cells of WT and APP-PS1 transgenic mice (inserts). c Besides co-expression of 5-Lox (green, using polyclonal 5-Lox antibody from Abcam #ab39347) in some microglia cells, also GFAP (red) positive astrocytes showed immunoreactivity for 5-Lox in WT and APP-PS1 mice (inserts). d FLAP (green, polyclonal FLAP antibody from Novus Biologicals #NBP1–84666) was ubiquitously expressed in Iba1 positive microglia cells (white) in WT and APP-PS1 transgenic mice. Dapi ( a - d ) was used as nucleus stain. Scale: 20 μm ( b - d right images), 50 μm ( a - d left images)

Article Snippet: 5-Lox (red, using polyclonal 5-Lox antibody from Abcam #ab39347) is expressed in FLAP (green, polyclonal FLAP antibody from Novus Biologicals NB300–891) positive cells associated with the amyloid plaque.

Techniques: Immunohistochemical staining, Expressing, Transgenic Assay, Staining

Journal: Acta Neuropathologica Communications

Article Title: Microglia depletion diminishes key elements of the leukotriene pathway in the brain of Alzheimer’s Disease mice

doi: 10.1186/s40478-020-00989-4

Figure Lengend Snippet: Immunohistochemical analysis of FLAP expression in the hippocampus. a Overall FLAP (green, polyclonal FLAP antibody from Novus Biologicals NBP1–84666) expression was more prominent in APP-PS1 compared to WT control animals and highly reduced with PLX5622 treatment. b FLAP co-localized with Iba1 (white) positive cells in all groups (arrows). Most interestingly, the intensity of FLAP staining in microglia at sites of amyloid plaques was reduced (red asterisk). Dapi was used as nucleus stain. Scale: 50 μm ( a ), 20 μm ( b )

Article Snippet: 5-Lox (red, using polyclonal 5-Lox antibody from Abcam #ab39347) is expressed in FLAP (green, polyclonal FLAP antibody from Novus Biologicals NB300–891) positive cells associated with the amyloid plaque.

Techniques: Immunohistochemical staining, Expressing, Control, Staining

Journal: Acta Neuropathologica Communications

Article Title: Microglia depletion diminishes key elements of the leukotriene pathway in the brain of Alzheimer’s Disease mice

doi: 10.1186/s40478-020-00989-4

Figure Lengend Snippet: 3D Render of images with Imaris Software. 5-Lox (red, using polyclonal 5-Lox antibody from Abcam #ab39347) is expressed in FLAP (green, polyclonal FLAP antibody from Novus Biologicals NB300–891) positive cells associated with the amyloid plaque. 5-Lox and FLAP are in close contact at sites of the nuclear membrane. Dapi was used as nucleus stain. Scale: 5 μm, 1 μm (insert left), 2 μm (insert right)

Article Snippet: 5-Lox (red, using polyclonal 5-Lox antibody from Abcam #ab39347) is expressed in FLAP (green, polyclonal FLAP antibody from Novus Biologicals NB300–891) positive cells associated with the amyloid plaque.

Techniques: Software, Membrane, Staining

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: Cells

Article Title: Mitochondrion-Mediated Cell Death through Erk1-Alox5 Independent of Caspase-9 Signaling.

doi: 10.3390/cells11193053

Figure Lengend Snippet: Figure 5. Alox5 and Alox5P in mediating caspase-9-independent cell death. (a–c) JMR cells trans- fected with non-targeting control siRNA (C) or individual siRNA targeting (a) Alox5 or (b) Alox5AP or (c) siRNA #1 targeting Alox5 and siRNA #1 targeting Alox5AP. The cells were treated with ABT-263, followed by quantitation of cell death. The cells were also used for Western blot analyses. (d) JMR cells transfected with siRNA targeting Alox5 or Alox5AP as in (c) were cultured with or without ABT-263, followed by staining with BODIPY 581/591 C11 and analyses by flow cytometry. Dashed line: unstained control. Data are presented as mean ± SD. Comparison with control siRNA group: ** p < 0.01.

Article Snippet: The following antibodies were used for Western blot: rabbit polyclonal or monoclonal antibodies to Alox5, ERK1, p38 MAPK, p44/42 MAPK (ERK1/2), phosphor-p44/p42 MAPK (p-ERK1/ERK2), RAD51, SAPK/JNK, SESN2 (Cell Signaling Technology), Alox5AP, DHODH, HADHA, MGST1, TIA-1 (Abcam), EndoG (ProSci), OXR1 (Bethyl Laboratory, Montgomery, TX, USA.) and PDP1 (Sigma); and mouse monoclonal antibodies to AIFM1, API5, PNKP (Santa Cruz Biotechnology), caspase-3, caspase-6 caspase-7, caspse-9, phosphor-p38 MAPK, phosphorSPAK/JNK (Cell Signaling Technology), FLAG (Sigma) and GFP (Origene).

Techniques: Control, Quantitation Assay, Western Blot, Transfection, Cell Culture, Staining, Cytometry, Comparison