Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.

doi: 10.4049/jimmunol.2200280

Figure Lengend Snippet: FIGURE 2. Inhibition of mTORC1 signaling in vitro decreased the phagocytic capacity of trout B cells. Representative flow cytometry dot plots of trout SPLs after treatment with DMSO or RAPA (100 nM) for 1 d in vitro incubated with 1-mm fluorescent latex beads and then stained with anti-trout IgT (A and B) or anti-IgM mAbs (C and D). “Low” indicates phagocytic IgT1 or IgM1 B cells internalizing one bead, and “High” represents phagocytic IgT1 or IgM1 B cells internalizing two or more beads. (E) Percentage of phagocytic B cells among total splenic IgT1 or IgM1 B cells after treatment with DMSO or RAPA (n 5 9 fish/group). (F) Percentage of high phagocytic B cells internalizing two and more beads among total splenic phagocytic IgT1 or IgM1 B cells after treatment with DMSO or RAPA (n 5 9 fish/group). Representative immunofluorescence microscopy of trout SPLs with DMSO or RAPA treatment in vitro incubated with 1-mm fluorescent latex beads (green; labeled with FITC) and then stained with anti-trout IgT (pink; G) or anti-trout IgM mAbs (red; H). Nuclei were stained with DAPI (blue). Black arrows point to internalized beads. Scale bars, 5 mm. Statistical differences were performed by paired Student t test. Data in (E) and (F) are representative of at least three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001. DIC, differential interference contrast; Phag−, nonphagocytic; Phag1, phagocytic.

Article Snippet: Thereafter, the PVDF membrane with intracellular proteins were incubated with antimTOR, anti-S6, anti-4EBP1, anti phospho-mTOR S2448, anti phospho-S6 Ser240/244, or anti phospho-4EBP1 Thr37/46 monoclonal rabbit Ab (Cell Signaling Technology) for 1 h. After washing four times, goat anti-rabbit IgG was added and incubated for 45 min at room temperature.

Techniques: Inhibition, In Vitro, Flow Cytometry, Incubation, Staining, Immunofluorescence, Microscopy, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.

doi: 10.4049/jimmunol.2200280

Figure Lengend Snippet: FIGURE 4. Inhibition of mTORC1 signaling in vivo decreased B cell survival and Ig-secreting capacities. Immunoblotting analysis showing total protein or phosphorylation levels of the indicated mTORC1 components containing mTOR (A), S6 (C), and 4EBP1 (E) in sorted IgT1 (left panel) and IgM1 (right panel) B cells from trout injected with DMSO or RAPA for 7 d. b-Actin was used as a loading control. The relative ratio of phosphorylation levels to those of total protein levels of mTOR (B), S6 (D), and 4EBP1 (F) in sorted B cells after treatment with DMSO or RAPA, evaluated by densitometric analysis of immunoblots from (A), (C), and (E), respectively (n 5 9 fish/group). (G) Flow cytometry analysis of SPLs from trout injected with DMSO (left panel) or RAPA (right panel) for 7 d and stained with anti-IgT and anti-IgM mAbs. Numbers adjacent to outlined boxes indicate the percentage of IgM1 (top left) and IgT1 (bottom right) B cells in the lymphocyte gate, respectively. (H) Percentage of IgT1 or IgM1 B cells among lymphocytes of spleen from trout injected with DMSO or RAPA (n 5 9 fish/group). Representative flow cytometry dot plots showing apoptotic IgT1 (I) and IgM1 (J) B cells among lymphocytes of spleen from trout injected with DMSO (left panel) or RAPA (right panel) for 7 d. (K) The relative ratio of apoptotic IgT1 or IgM1 B cells in total number of RAPA injection group IgT1 or IgM1 B cells to DMSO injection group (n 5 9 fish/group). Representative flow cytometry dot plot showing proliferation of IgT1 (L) and IgM1 (M) B cells among lymphocytes of spleen from fish injected with DMSO (left panel) or RAPA (right panel) for 7 d. (N) Percentage of EdU1 cells among the splenic IgT1 or IgM1 B cell populations in fish injected with DMSO or RAPA (n 5 9 fish/group). (O) Immunoblotting analysis of IgT, IgM, and IgD in supernatants of spleen explant from fish injected with DMSO (left panel) or RAPA (right panel) for 7 d. (P) The relative ratio of IgT, IgM, and IgD secretion in the RAPA group to those of the DMSO group, evaluated by densitometric analysis of immunoblots from (O) (n 5 10 fish/group). Statistical differences were performed by unpaired Student t test. Data in (B), (D), (F), (H), (K), (N), and (P) are representative of at least three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Thereafter, the PVDF membrane with intracellular proteins were incubated with antimTOR, anti-S6, anti-4EBP1, anti phospho-mTOR S2448, anti phospho-S6 Ser240/244, or anti phospho-4EBP1 Thr37/46 monoclonal rabbit Ab (Cell Signaling Technology) for 1 h. After washing four times, goat anti-rabbit IgG was added and incubated for 45 min at room temperature.

Techniques: Inhibition, In Vivo, Western Blot, Phospho-proteomics, Injection, Control, Flow Cytometry, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.

doi: 10.4049/jimmunol.2200280

Figure Lengend Snippet: FIGURE 6. RAPA treatment in fish significantly decreased IgM-mediated bacterial agglutination and increased bacterial load and fish mortalities upon F. columnare reinfection. (A) Scheme of the experiment of IgM depletion in serum strategy. Magnetic protein G beads were firstly incubated with anti-trout IgM mAb in 4◦C for 2 h to generate protein G coated with anti-IgM mAb. IgM from serum of the 28DPI-S-DMSO group was depleted by incubating this serum with protein G beads coated with anti-IgM mAbs. Thereafter, F. columnare was incubated with serum from naive fish or the 28DPI-S-DMSO, 28DPI- S-RAPA, and 28DPI-S-DMSO-IgMDEP group in 4◦C. The agglutination of F. columnare in different groups was analyzed at 10 min after coincubation, and the growth of F. columnare was detected at 2 h after coincubation (n 5 6 fish/group). (B) Representative immunofluorescence images of GFPF. columnare (green; OD540 5 0.5, ∼106 CFU/ml) agglutination after incubating for 10 min with serum (1:10 dilution) from fish in naive, 28DPI-S-DMSO, 28DPI-S- RAPA, and 28DPI-S-DMSO-IgMDEP groups. The top and bottom lines of images are representative of one observed field obtained from three to six obser- vations from three independent slides. Scale bars, 10 mm. (C) The growth curve of F. columnare (initial OD540 5 0.3) incubated with serum (1:10 dilution) from fish in the naive, 28DPI-S-DMSO, 28DPI-S-DMSO-IgMDEP, and 28DPI-S-RAPA groups (n 5 9 fish/group). (D) The OD540 value of F. columnare after incubating with serum (1:10 dilution) from fish in the naive, 28DPI-S-DMSO, 28DPI-S-DMSO-IgMDEP, and 28DPI-S-RAPA groups for 15, 18, and 21 h (n 5 9 fish/group). Different letters (a, b, and c) represent that a significant difference (p < 0.05) exists in these groups within each time. Statistical differences were performed by one-way ANOVA. Data in (C) and (D) are representative of at least three independent experiments (mean ± SEM).

Article Snippet: Thereafter, the PVDF membrane with intracellular proteins were incubated with antimTOR, anti-S6, anti-4EBP1, anti phospho-mTOR S2448, anti phospho-S6 Ser240/244, or anti phospho-4EBP1 Thr37/46 monoclonal rabbit Ab (Cell Signaling Technology) for 1 h. After washing four times, goat anti-rabbit IgG was added and incubated for 45 min at room temperature.

Techniques: Agglutination, Incubation, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: Isolation of Hyperactive Mutants of Mammalian Target of Rapamycin

doi: 10.1074/jbc.m801546200

Figure Lengend Snippet: FIGURE 2. mTOR mutants exhibit enhanced kinase activity in vitro. HeLa cellsweretransfectedwiththeexpressionplasmidsforwild-typeFLAG-mTOR (WT), FLAG-mTORSL1 (SL1), FLAG-mTORI2017T (I2017T), and FLAG-mTORSL1IT (SL1IT). After 48 h transfection cells were lysed with CHAPS buffer, and FLAG-mTOR and its variants were immunoprecipitated (IP) with anti-FLAG beads. The kinase reaction was performed as described under “Experimental Procedures.”ThephosphorylationlevelsandtheamountsofGST-4EBP1were examined by immunoblotting using an anti-phospho-4EBP1(Thr-37/46) and by Coomassie Brilliant Blue (CBB) staining, respectively.

Article Snippet: Antibodies for phospho-S6K1 Thr-389, phospho-4EBP1 Thr-37/46, phospho-Akt Ser-473, Raptor, mLst8, and 4EBP1 were from Cell Signaling Technology (Beverly,MA).Anti-S6K1 antibodywas fromSanta Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, In Vitro, Transfection, Immunoprecipitation, Western Blot, Staining

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs

doi: 10.3724/abbs.2022159

Figure Lengend Snippet: Intervening with 4E-BP1/eIF4E-dependent translation initiation is not able to rescue the decreased PTEN mediated by mTOR inhibition (A,C) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs after treatment with 10 nM rapamycin for 24 h after 4E-BP1 knockdown. (B,D) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs after treatment with 10 nM rapamycin for 24 h after eIF4E knockdown.

Article Snippet: The details of the primary antibodies were as follows: PTEN (9559, 1:1000; CST, Shanghai, China), 4E-BP1 (9644, 1:1000; CST), P-4E-BP1 T37/46 (2855, 1:1000; CST), S6 (2217, 1:1000; CST), P-S6 Ser235/236 (4858, 1:1000; CST), AKT1 (2938, 1:1000; CST), P-AKT1 S473 (9018, 1:1000), TSC2 (4308, 1:1000; CST), Lamin A/C (4777, 1:1000; CST), GAPDH (97166, 1:1000; CST), eIF4E (2067, 1:1000; CST), P62 (8025, 1:1000; CST), β-catenin (9587S, 1:1000; CST), and β-actin (SC-47778, 1:3000; Santa Cruz, Shanghai, China).

Techniques: Inhibition, Knockdown