Journal: Cell reports

Article Title: Antigen-Presenting Cell-Intrinsic PD-1 Neutralizes PD-L1 in cis to Attenuate PD-1 Signaling in T Cells

doi: 10.1016/j.celrep.2018.06.054

Figure Lengend Snippet: (A) Left: cartoon showing a cell culture assay in which PD-1 transduced Jurkat cells were stimulated with three types of antigen-loaded Raji B cells: (1) parental Raji cells that express neither PD-L1 nor PD-1, (2) Raji cells transduced with only PD-L1 (PD-L1+), and (3) Raji transduced with both PD-L1 and PD-1 (PD-L1+/PD-1+). On the right are FACS histograms showing PD-1 and PD-L1 surface expression of parental Jurkat cells, PD-1–mGFP transduced Jurkat (PD-1+) cells, parental Raji cells, PD-L1–mCherry transduced Raji (PD-L1+) cells, and Raji cells co-transduced with both PD-1–mGFP and PD-L1–mCherry (PD-L1+/PD-1+). a.u., arbitrary units. (B–D) Jurkat cells expressing PD-1–mGFP (PD-1+) were conjugated with Raji B cells (parental) (B), Raji cells transduced with only PD-L1–mCherry (PD-L1+) (C), or Raji cells co-transduced with PD-L1–mCherry and unlabeled PD-1 (PD-L1+/PD-1+) (D), as illustrated in the cartoon on the left (CD28 and B7 are omitted in this cartoon for simplicity). Shown on the right are confocal images of the cell conjugate acquired 2 min after cell-cell contact. mGFP and mCherry signals are shown as green and magenta, respectively. Scale bars, 10 µm. (E) Bar graph summarizing the interface enrichment indices (calculated as described in STAR Methods) of the three conditions shown in (B). Data are shown as mean ± SEM; n = 35 cells from three independent experiments. See also . (F) A representative western blot showing the levels ZAP70-Y493 phosphorylation and p85 (PI3K regulatory subunit) co-immunoprecipitated (IP) with CD28 from the lysates of the indicated Jurkat-Raji co-culture. Jurkat cells expressing PD-1–mGFP (PD-1+) were stimulated with Raji cells (parental), Raji transduced with PD-L1–mCherry (PD-L1+), or Raji cells co-transduced with PD-L1–mCherry and PD-1–mGFP (PD-L1+/PD-1+); the times at which the co-culture was lysed are indicated (STAR Methods). WCL, whole cell lysate. (G and H) Bar graphs summarizing immunoblots in (F), including pY493-ZAP70 immunoblot (G) and CD28 coIP p85 immunoblot (H). The optical density corresponding to each band was quantified by ImageJ and normalized to the conditions in which parental Raji cells were used as the APCs. Data are presented as mean ± SEM from three independent replicates. (I) Bar graph summarizing IL-2 levels in the medium of the indicated Jurkat-Raji co-culture, as described in (B)–(D), 24 hr after cell-cell contact. Data are presented as mean ± SEM from three independent measurements, with each run performed in triplicate.

Article Snippet: ZAP70 pY493 antibody , Cell Signaling Technology , Cat # 2704S; RRID: AB_2217457.

Techniques: Cell Culture, Transduction, Expressing, Western Blot, Phospho-proteomics, Immunoprecipitation, Co-Culture Assay

Journal: Cell reports

Article Title: Antigen-Presenting Cell-Intrinsic PD-1 Neutralizes PD-L1 in cis to Attenuate PD-1 Signaling in T Cells

doi: 10.1016/j.celrep.2018.06.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ZAP70 pY493 antibody , Cell Signaling Technology , Cat # 2704S; RRID: AB_2217457.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Software, Imaging

Journal: Cell Reports Medicine

Article Title: Dysregulation of FLVCR1a-dependent mitochondrial calcium handling in neural progenitors causes congenital hydrocephalus

doi: 10.1016/j.xcrm.2024.101647

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal, anti-GRP75 , Proteintech, Rosemont, IL, USA , 14887-1-AP; RRID:AB_2120458.

Techniques: Control, Purification, Marker, Recombinant, Sterility, Protease Inhibitor, Bicinchoninic Acid Protein Assay, ATP Bioluminescent Assay, Reverse Transcription, Transfection, Imaging, SYBR Green Assay, Plasmid Preparation, shRNA, Software, Variant Assay

Journal: Circulation

Article Title: Cardiac Raptor Ablation Impairs Adaptive Hypertrophy, Alters Metabolic Gene Expression, and Causes Heart Failure in Mice

doi: 10.1161/circulationaha.110.977066

Figure Lengend Snippet: Figure 1. Functional, molecular, and metabolic analyses of raptor-cKO and control mice reveal no differences at 2 weeks after tamoxifen. Adult male MHC-MerCreMer/raptorfl/fland con- trol mice were injected with tamoxifen or vehicle for 5 days and euthanized 2 weeks later for analysis by Western blotting (A and B), real-time polymerase chain reaction (C), or ex vivo isolated heart experiments (D and E). For A and B, cardiac (20 g) or skeletal (10 g) muscle proteins were separated by SDS-PAGE and probed with antibodies as indicated. Wild-type-1 (WT-1) indicates vehicle/MHC-MerCreMer/raptorfl/fl; WT-2, tamoxifen/ MHC-MerCreMer/raptor/; cKO, tamoxifen/MHC-MerCreMer/ raptorfl/fl; n3 to 5 per group. For C, 1-way ANOVA analysis resulted in values of P0.90, 0.42, 0.86, 0.81, 0.98, and 0.72 for atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), peroxisome proliferator-activated receptor (PPAR), PGC1, GLUT1, and GLUT4, respectively. HW indicates heart weight; 4E-BP1, 4E-binding protein 1.

Article Snippet: Polyclonal rabbit antibodies against phospho-Akt (Ser473), phospho-Akt (Thr308), Akt total, GSK3β (Ser9), phospho-4E-BP1 (Thr70), 4E-BP1 total, phospho-S6 kinase (Ser240/244), p70 S6 kinase, mTOR and monoclonal antibody against raptor were all from Cell Signaling Technology (Danvers, MA).

Techniques: Functional Assay, Control, Injection, Western Blot, Real-time Polymerase Chain Reaction, Ex Vivo, Isolation, SDS Page, Binding Assay

Journal: Circulation

Article Title: Cardiac Raptor Ablation Impairs Adaptive Hypertrophy, Alters Metabolic Gene Expression, and Causes Heart Failure in Mice

doi: 10.1161/circulationaha.110.977066

Figure Lengend Snippet: Figure 4. Effect of raptor deletion on molecules that regulate protein synthesis (A through D) and on gene expression of E3 enzymes involved in proteasomal degradation (E) at 1 week after surgery with groups as in Figures 2 and 3. A, Representative immunoblots of cardiac protein extracts (20 g). Equal loading was verified with Ponceau C, and membranes probed with specific antibodies to indi- cated proteins. For B through D, labeled bands were quantified with LI-COR Odyssey imaging software (n6 per group). For E, total RNA was used to determine relative mRNA levels by quantitative polymerase chain reaction with primers as listed in the online-only Data Supplement (n4 per group). mTOR indicates mammalian target of rapamycin; 4E-BP1, 4E-binding protein 1. ###P0.001, #P0.05 for transverse aortic constriction (TAC) vs sham; ***P0.001, **P0.01, *P0.05 for raptor-cKO vs wild-type.

Article Snippet: Polyclonal rabbit antibodies against phospho-Akt (Ser473), phospho-Akt (Thr308), Akt total, GSK3β (Ser9), phospho-4E-BP1 (Thr70), 4E-BP1 total, phospho-S6 kinase (Ser240/244), p70 S6 kinase, mTOR and monoclonal antibody against raptor were all from Cell Signaling Technology (Danvers, MA).

Techniques: Gene Expression, Western Blot, Labeling, Imaging, Software, Real-time Polymerase Chain Reaction, Binding Assay