tor/mtor antibody  (Bio-Techne corporation)

Journal: Frontiers in Aging Neuroscience

Article Title: The APOE ε3/ε4 Genotype Drives Distinct Gene Signatures in the Cortex of Young Mice

doi: 10.3389/fnagi.2022.838436

Figure Lengend Snippet: Creation and validation of APOE allelic series. (A) Schematic of human APOE allele insertion at the mouse Apoe locus on chromosome 7. Exon 1 remained unchanged (mouse) and exons 2–4 were replaced with human APOE ε4 sequence through homology directed repair (HDR). The neomycin cassette was removed (* indicated by asterisk) through Flp-Frt breeding (Flp-Frt sites not shown). The APOE ε3 SNP was changed through CRISPR mediated endonuclease activity. Critical amino acids differing between APOE ε4 and APOE ε3 noted in red. (B) PCR based assay to identify humanized APOE insertions at exon 2 vs. WT mouse Apoe (no insertion) at exon 2. (C) Sanger sequencing to distinguish APOE ε3 and/or APOE ε4 alleles. (D) Western blotting revealed significant differences in APOE ε4 levels between genotypes.

Article Snippet: Antibodies used include: Pan human APOE (AB947, Millipore, Burlington, MA, United States); Mouse Apoe (NB100-240, Novus Biologicals, Centennial, CO, United States); human APOE ε4 (NBP1-49529, Novus Biologicals); Actin (ab179467, Abcam, Cambridge, United Kingdom).

Techniques: Biomarker Discovery, Sequencing, CRISPR, Activity Assay, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: The APOE ε3/ε4 Genotype Drives Distinct Gene Signatures in the Cortex of Young Mice

doi: 10.3389/fnagi.2022.838436

Figure Lengend Snippet: Cortical transcriptome analysis revealed unique functional enrichment for the APOE ε3/ε4 genotype at 2 months. (A) PCA showed distinct clusters of male and female samples. (B) PCA showed samples do not cluster by APOE genotype at two mos. (C) Number of significant genes for each linear model predictor term ( p < 0.05). (D) Number of significant genes unique to 2-month APOE ε4/ε4 and APOE ε4/ε4 when compared to APOE ε3/ε3 , as well as the number of significant genes intersecting both lists. (E) Functional enrichment of the 360 significant genes ( p < 0.10) unique to APOE ε3/ε4 compared to APOE ε3/ε3 . (F) Percentage of genes expressed by cell type according to the BrainRNAseq dataset.

Article Snippet: Antibodies used include: Pan human APOE (AB947, Millipore, Burlington, MA, United States); Mouse Apoe (NB100-240, Novus Biologicals, Centennial, CO, United States); human APOE ε4 (NBP1-49529, Novus Biologicals); Actin (ab179467, Abcam, Cambridge, United Kingdom).

Techniques: Functional Assay

Journal: Frontiers in Aging Neuroscience

Article Title: The APOE ε3/ε4 Genotype Drives Distinct Gene Signatures in the Cortex of Young Mice

doi: 10.3389/fnagi.2022.838436

Figure Lengend Snippet: Significant genes unique for APOE ε3/ε4 genotype showed functional enrichment for ‘extracellular matrix’ and ‘response to wounding’ at 4 months. (A,B) Average number of rotations per night during the dark cycle for female (A) and males (B) across all APOE genotypes for the six mice per group that were selected for RNA-seq. Mean is representative of all mice, including those that were not sequenced (see also ). (C) Number of significant genes per each linear model predictor term. Red arrows highlight APOE ε4/ε4 and APOE ε3/ε4 terms used in panel (D) . (D) Number of significant genes unique to APOE ε3/ε4 when compared to APOE ε3/ε3 , and APOE ε4/ε4 compared to APOE ε3/ε3 , as well as the number of significant genes intersecting both groups. (E) Functional enrichment of the 253 genes unique to APOE ε3/ε4 compared to APOE ε3/ε3 (left), 374 genes unique to APOE ε4/ε4 compared to APOE ε3/ε3 (right) and 74 intersection genes between the two groups (middle) ( p < 0.05).

Article Snippet: Antibodies used include: Pan human APOE (AB947, Millipore, Burlington, MA, United States); Mouse Apoe (NB100-240, Novus Biologicals, Centennial, CO, United States); human APOE ε4 (NBP1-49529, Novus Biologicals); Actin (ab179467, Abcam, Cambridge, United Kingdom).

Techniques: Functional Assay, RNA Sequencing

Journal: Biomolecules

Article Title: Calystegines Improve the Metabolic Activity of Human Adipose Derived Stromal Stem Cells (ASCs) under Hyperglycaemic Condition through the Reduction of Oxidative/ER Stress, Inflammation, and the Promotion of the AKT/PI3K/mTOR Pathway

doi: 10.3390/biom12030460

Figure Lengend Snippet: List of antibodies employed for protein profiling using western blot analysis.

Article Snippet: mTOR , 1:500 , Novus Biologicals, nb100-240.

Techniques: Western Blot

Journal: Autophagy

Article Title: Induction of autophagy and suppression of type I IFN secretion by CSFV

doi: 10.1080/15548627.2020.1739445

Figure Lengend Snippet: The suppression of MTOR activity is associated with CSFV-mediated autophagy. (A and B) PK-15 (A) and 3D4/2 (B) cells were infected with CSFV at an MOI of 1 or mock-infected. At 6, 12 and 24 hpi, cell samples were analyzed by immunoblotting with antibodies against p-MTOR, LC3B, CSFV-E2, and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (C) PK-15 and 3D4/2 cells were mock-infected or infected with CSFV (MOI = 1) or treated with 3-MA (5 mM) or rapamycin (Rapa, 100 nM) for 48 h. The level of protein was quantified using Image-Pro Plus. Error bars: mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (D) PK-15 and 3D4/2 cells were pretreated with rapamycin (100 nM) or DMSO (control) for 1.5 h, followed by CSFV adsorption for 1.5 h at an MOI of 1. The host cells were further cultured in fresh medium in the presence of rapamycin (100 nM). At 24 and 48 hpi, the mRNA level of Npro(CSFV) were detected by qRT-PCR as described in Materials and Methods. (E) PK-15 and 3D4/2 cells were pretreated with 3-MA (5 mM) or DMSO (control) for 1.5 h, followed by CSFV adsorption for 1.5 h at an MOI of 1. The host cells were further cultured in fresh medium in the presence of 3-MA (5 mM). At 24 and 48 hpi, the mRNA level of Npro(CSFV) were detected by qRT-PCR as described in Materials and Methods. (F) Analysis of autophagy-related proteins by western blotting in infected CSFV splenic cells. splenic lysates were prepared as described in Materials and Methods, splenic samples were analyzed by immunoblotting with antibodies against p-MTOR, p-PRKAA, CAMKK2, MAPK1/3, p-MAPK1/3, p-AKT, AKT, and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA)

Article Snippet: The primary antibodies were specific for p-PRKAA/AMPKα (Thr172) (Cell Signaling Technology, 2535), p-MAPK1/3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology, 9101), MAPK1/3 (ERK1/2; Cell Signaling Technology, 9102), AKT (Cell Signaling Technology, 9272), p-AKT (Ser473; Cell Signaling Technology, 9272), PRKAA/AMPKα (Novus Biologicals, NB100-239), MTOR (Novus Biologicals, NB100-240), BECN1/Beclin 1 (Novus Biologicals, NB110-87,318), CAMKK2/CaMKKβ (Abcam ab168818), LC3B (Novus Biologicals, NB100-2220), p-MTOR (S2448; Abcam, ab109268), HSP90AB1 (Proteintech, 11,405-1-AP), MAVS (Proteintech, 14,341-1-AP), Flag (Beyotime, AF519), HA (Beyotime, AH158), GFP (Beyotime, AF1483), and GAPDH (Gene Tex, GTX100118), Tubulin (Beyotime, AT819), ACTB (Beyotime, AA128).

Techniques: Activity Assay, Infection, Western Blot, Control, Adsorption, Cell Culture, Quantitative RT-PCR

Journal: Autophagy

Article Title: Induction of autophagy and suppression of type I IFN secretion by CSFV

doi: 10.1080/15548627.2020.1739445

Figure Lengend Snippet: CSFV dysregulates AKT-MTOR signaling in 3D4/2 and PK-15 cells to induce autophagy. (A) 3D4/2 and PK-15 cells were infected with CSFV at an MOI of 1 or mock-infected. At 6, 12 and 24 hpi, cell samples were analyzed by immunoblotting with antibodies against p-AKT and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (B) 3D4/2 and PK-15 cells were pretreated with INS (100 ng/ml) or DMSO (control) for 24 h, and cell samples were analyzed by immunoblotting with antibodies against p-AKT and ACTB (loading control). (C and D) PK-15 (C) and 3D4/2 (D) cells were pretreated with INS (100 ng/ml) or DMSO (control) for 1.5 h, followed by mock infection and CSFV infection for 1.5 h at an MOI of 1. The host cells were cultured in fresh medium in the presence or absence of INS (100 ng/ml). At 36 hpi, cell samples were analyzed by immunoblotting with antibodies against p-MTOR, LC3B, AKT, p-AKT, CSFV-E2, and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (E) 3D4/2 and PK-15 cells were pretreated and infected as described in (C and D). At 48 hpi, both the intracellular and extracellular viral titers were measured by TCID50. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (F) PK-15 cells were pretreated and infected as described in (C). At 48 hpi, the cells were fixed and processed for indirect immunofluorescence using antibodies against LC3B, followed by the corresponding secondary antibodies conjugated to Alexa Fluor 488 as described in the Materials and Methods. The LC3 fluorescence signals were analyzed by confocal immunofluorescence microscopy. In the images, LC3 staining is shown in green. Scale bar: 25 µm. The average number of LC3 puncta per cell from at least 20 cells in each group. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (G and H) PK-15 (G) and 3D4/2 (H) cells were pretreated with INS (100 ng/ml) or DMSO (control) for 1.5 h, followed by CSFV adsorption for 1.5 h at an MOI of 1. The host cells were further cultured in fresh medium in the presence of INS (100 ng/ml) or DMSO (control). At 24 and 48 hpi, the mRNA level of Npro (CSFV) were detected by qRT-PCR as described in Materials and Methods

Article Snippet: The primary antibodies were specific for p-PRKAA/AMPKα (Thr172) (Cell Signaling Technology, 2535), p-MAPK1/3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology, 9101), MAPK1/3 (ERK1/2; Cell Signaling Technology, 9102), AKT (Cell Signaling Technology, 9272), p-AKT (Ser473; Cell Signaling Technology, 9272), PRKAA/AMPKα (Novus Biologicals, NB100-239), MTOR (Novus Biologicals, NB100-240), BECN1/Beclin 1 (Novus Biologicals, NB110-87,318), CAMKK2/CaMKKβ (Abcam ab168818), LC3B (Novus Biologicals, NB100-2220), p-MTOR (S2448; Abcam, ab109268), HSP90AB1 (Proteintech, 11,405-1-AP), MAVS (Proteintech, 14,341-1-AP), Flag (Beyotime, AF519), HA (Beyotime, AH158), GFP (Beyotime, AF1483), and GAPDH (Gene Tex, GTX100118), Tubulin (Beyotime, AT819), ACTB (Beyotime, AA128).

Techniques: Infection, Western Blot, Control, Cell Culture, Immunofluorescence, Fluorescence, Microscopy, Staining, Adsorption, Quantitative RT-PCR

Journal: Autophagy

Article Title: Induction of autophagy and suppression of type I IFN secretion by CSFV

doi: 10.1080/15548627.2020.1739445

Figure Lengend Snippet: PRKAA is an upstream switch of MTOR in CSFV-mediated autophagy. (A) 3D4/2 and PK-15 cells were infected with CSFV at an MOI of 1 or mock-infected. At 24 and 48 hpi, cell samples were analyzed by immunoblotting with antibodies against p-PRKAA and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (B) 3D4/2 and PK-15 cells were pretreated with compound C (50 µM) or DMSO (control) for 24 h, and cell samples were analyzed by immunoblotting with antibodies against p-PRKAA and ACTB (loading control). (C and D) PK-15(C) and 3D4/2(D) cells were pretreated with compound C (50 µM) or DMSO (control) for 1.5 h after 1.5 h of virus absorption at an MOI of 1 and mock infection. The host cells were further cultured in fresh medium in the presence or absence of compound C (50 µM). At 36 hpi, cell samples were analyzed by immunoblotting with antibodies against p-MTOR, LC3B, PRKAA, p-PRKAA, CAMKK2, CSFV-E2, and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (E and I) 3D4/2 and PK-15 cells were pretreated and infected as described in (C, D, and H). At 48 hpi, both the intracellular and extracellular viral titers were measured by TCID50. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (F) PK-15 cells were pretreated and infected as described in (C). At 48 hpi, cells were analyzed as described in the legend to Figure 2F. (G) PK-15 cells were transfected with negative control shRNA or specific shRNA against PRKAA for 48 h and then analyzed by western blotting. (H) PK-15 cells were transfected with either shNC or PRKAA-specific shRNA for 6 h and then infected with CSFV or mock-infected for an additional 48 h, after which cells were harvested for western blotting assay

Article Snippet: The primary antibodies were specific for p-PRKAA/AMPKα (Thr172) (Cell Signaling Technology, 2535), p-MAPK1/3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology, 9101), MAPK1/3 (ERK1/2; Cell Signaling Technology, 9102), AKT (Cell Signaling Technology, 9272), p-AKT (Ser473; Cell Signaling Technology, 9272), PRKAA/AMPKα (Novus Biologicals, NB100-239), MTOR (Novus Biologicals, NB100-240), BECN1/Beclin 1 (Novus Biologicals, NB110-87,318), CAMKK2/CaMKKβ (Abcam ab168818), LC3B (Novus Biologicals, NB100-2220), p-MTOR (S2448; Abcam, ab109268), HSP90AB1 (Proteintech, 11,405-1-AP), MAVS (Proteintech, 14,341-1-AP), Flag (Beyotime, AF519), HA (Beyotime, AH158), GFP (Beyotime, AF1483), and GAPDH (Gene Tex, GTX100118), Tubulin (Beyotime, AT819), ACTB (Beyotime, AA128).

Techniques: Infection, Western Blot, Control, Virus, Cell Culture, Transfection, Negative Control, shRNA

Journal: Autophagy

Article Title: Induction of autophagy and suppression of type I IFN secretion by CSFV

doi: 10.1080/15548627.2020.1739445

Figure Lengend Snippet: CSFV induces autophagy through the CAMKK2-PRKAA-MTOR signaling pathway. (A) 3D4/2 and PK-15 cells were infected with CSFV at an MOI of 1 or mock-infected. At 6, 12 and 24 hpi, cell samples were analyzed by immunoblotting with antibodies against CAMKK2 and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (B) 3D4/2 and PK-15 cells were pretreated with STO-609 (50 µM) or DMSO (control) for 24 h, and cell samples were prepared and analyzed by immunoblotting with antibodies against CAMKK2 and ACTB (loading control). (C and D) PK-15 (C) and 3D4/2 (D) cells were pretreated with STO-609 (50 µM) or DMSO (control) for 1.5 h, followed by mock infection and CSFV infection for 1.5 h at an MOI of 1. The host cells were further cultured in fresh medium in the presence or absence of STO-609 (50 µM). At 36 hpi, cell lysates were prepared and analyzed by immunoblotting using anti-p-MTOR, anti-LC3B, anti-PRKAA, anti-p-PRKAA, anti-CAMKK2, anti-E2, and anti-ACTB antibodies. The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (E) 3D4/2 and PK-15 cells were pretreated and infected as described in (C and D). At 48 hpi, both the intracellular and extracellular viral titers were measured by TCID50. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (F) PK-15 cells were pretreated and infected as described in (C). At 48 hpi, cells were analyzed as described in the legend to Figure 2F. (G and H) PK-15(G) and 3D4/2(H) cells were pretreated with STO-609 (50 µM) or DMSO (control) for 1.5 h, followed by CSFV adsorption for 1.5 h at an MOI of 1. The host cells were further cultured in fresh medium in the presence of STO-609 (50 µM). At 24 and 48 hpi, the mRNA level of Npro(CSFV) were detected by qRT-PCR as described in Materials and Methods

Article Snippet: The primary antibodies were specific for p-PRKAA/AMPKα (Thr172) (Cell Signaling Technology, 2535), p-MAPK1/3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology, 9101), MAPK1/3 (ERK1/2; Cell Signaling Technology, 9102), AKT (Cell Signaling Technology, 9272), p-AKT (Ser473; Cell Signaling Technology, 9272), PRKAA/AMPKα (Novus Biologicals, NB100-239), MTOR (Novus Biologicals, NB100-240), BECN1/Beclin 1 (Novus Biologicals, NB110-87,318), CAMKK2/CaMKKβ (Abcam ab168818), LC3B (Novus Biologicals, NB100-2220), p-MTOR (S2448; Abcam, ab109268), HSP90AB1 (Proteintech, 11,405-1-AP), MAVS (Proteintech, 14,341-1-AP), Flag (Beyotime, AF519), HA (Beyotime, AH158), GFP (Beyotime, AF1483), and GAPDH (Gene Tex, GTX100118), Tubulin (Beyotime, AT819), ACTB (Beyotime, AA128).

Techniques: Infection, Western Blot, Control, Cell Culture, Adsorption, Quantitative RT-PCR

Journal: Autophagy

Article Title: Induction of autophagy and suppression of type I IFN secretion by CSFV

doi: 10.1080/15548627.2020.1739445

Figure Lengend Snippet: CSFV-induced [Ca2+]cyto increase potently induces autophagy through the CAMKK2-PRKAA-MTOR signaling pathway. (A) 3D4/2 and PK-15 cells were pretreated with BAPTA-AM (18 µM) or DMSO (control) for 24 h, and cell samples were analyzed by immunoblotting with antibodies against CAMKK2 and ACTB (loading control). (B and C) PK-15 (B) and 3D4/2 (C) cells were pretreated with ionomycin (7 µM), BAPTA-AM (18 µM) or DMSO (control) for 1.5 h after 1.5 h of virus absorption at an MOI of 1 and mock infection. The host cells were further cultured in fresh medium in the presence or absence of ionomycin (7 µM), BAPTA-AM (18 µM). At 36 hpi, the expression of p-MTOR, LC3B, PRKAA, p-PRKAA, CAMKK2, CSFV-E2, and ACTB (loading control) were analyzed by immunoblotting. The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (D) PK-15 cells were pretreated and infected as described in (B). At 48 hpi, cells were analyzed as described in the legend to Figure 2F. (E) PK-15 cells were pretreated and infected as described in (B). At 48 hpi, cells were stained with BBcellProbeTM F04 solution at 37°C for 30 min. The fluorescence signals were visualized with a fluorescence microscope. The IOD/area of fluorescence signals was determined in each group. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (F) 3D4/2 and PK-15 cells were pretreated and infected as described in (B and C). At 48 hpi, both the intracellular and extracellular viral titers were measured by TCID50. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (G) PK-15 and 3D4/2 cells were pretreated with BAPTA-AM (18 µM) or DMSO (control) for 1.5 h, followed by CSFV adsorption for 1.5 h at an MOI of 1. The host cells were further cultured in fresh medium in the presence of BAPTA-AM (18 µM). At 24 and 48 hpi, the mRNA level of Npro(CSFV) were detected by qRT-PCR as described in Materials and Methods

Article Snippet: The primary antibodies were specific for p-PRKAA/AMPKα (Thr172) (Cell Signaling Technology, 2535), p-MAPK1/3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology, 9101), MAPK1/3 (ERK1/2; Cell Signaling Technology, 9102), AKT (Cell Signaling Technology, 9272), p-AKT (Ser473; Cell Signaling Technology, 9272), PRKAA/AMPKα (Novus Biologicals, NB100-239), MTOR (Novus Biologicals, NB100-240), BECN1/Beclin 1 (Novus Biologicals, NB110-87,318), CAMKK2/CaMKKβ (Abcam ab168818), LC3B (Novus Biologicals, NB100-2220), p-MTOR (S2448; Abcam, ab109268), HSP90AB1 (Proteintech, 11,405-1-AP), MAVS (Proteintech, 14,341-1-AP), Flag (Beyotime, AF519), HA (Beyotime, AH158), GFP (Beyotime, AF1483), and GAPDH (Gene Tex, GTX100118), Tubulin (Beyotime, AT819), ACTB (Beyotime, AA128).

Techniques: Control, Western Blot, Virus, Infection, Cell Culture, Expressing, Staining, Fluorescence, Microscopy, Adsorption, Quantitative RT-PCR

Journal: Autophagy

Article Title: Induction of autophagy and suppression of type I IFN secretion by CSFV

doi: 10.1080/15548627.2020.1739445

Figure Lengend Snippet: NS5A induces autophagy through the activation of the CAMKK2-PRKAA-MTOR signaling pathway. (A) 3D4/2 and PK-15 cells were transfected with p-EGFP-Erns (CSFV), p-EGFP-E1 (CSFV), p-EGFP-E2 (CSFV), p-EGFP-C (CSFV), and p-EGFP-NS5A (CSFV) vectors. At 48 hpi, cell samples were analyzed by immunoblotting with antibodies against p-MTOR, PRKAA, p-PRKAA, CAMKK2, LC3B, and ACTB (loading control), and the efficiency of transfection was measured using an antibody against GFP. The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (B) 3D4/2 and PK-15 cells were transfected with p-EGFP-Npro (CSFV), p-EGFP- NS4A (CSFV), p-EGFP-NS2 (CSFV), p-EGFP- NS5A (CSFV), p-EGFP-NS3 (CSFV), and p-EGFP-C1 vectors. At 48 hpi, cell samples were analyzed by immunoblotting with antibodies against p-MTOR, PRKAA, p-PRKAA, CAMKK2, and ACTB (loading control), and the efficiency of transfection was measured using an antibody against GFP. The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (C) 3D4/2 and PK-15 cells were transfected with either pEGFP-C1 or pEGFP-NS5A. At 24 and 48 hpi, the cell samples were analyzed by immunoblotting with antibodies against p-MTOR, p-PRKAA, CAMKK2, MAPK1/3, p-MAPK1/3, LC3B, p-AKT, AKT, and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (D) PK-15 cells were transfected with plasmids encoding NS5A-Flag and HSP90AB1-HA protein, followed by immunoprecipitation (IP) with anti-Flag beads and immunoblot analysis with anti-HA. (E) PK-15 cells were transfected with plasmids encoding NS5A-Flag protein, followed by immunoprecipitation (IP) with anti-HSP90AB1 antibody and immunoblot analysis with anti-Flag

Article Snippet: The primary antibodies were specific for p-PRKAA/AMPKα (Thr172) (Cell Signaling Technology, 2535), p-MAPK1/3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology, 9101), MAPK1/3 (ERK1/2; Cell Signaling Technology, 9102), AKT (Cell Signaling Technology, 9272), p-AKT (Ser473; Cell Signaling Technology, 9272), PRKAA/AMPKα (Novus Biologicals, NB100-239), MTOR (Novus Biologicals, NB100-240), BECN1/Beclin 1 (Novus Biologicals, NB110-87,318), CAMKK2/CaMKKβ (Abcam ab168818), LC3B (Novus Biologicals, NB100-2220), p-MTOR (S2448; Abcam, ab109268), HSP90AB1 (Proteintech, 11,405-1-AP), MAVS (Proteintech, 14,341-1-AP), Flag (Beyotime, AF519), HA (Beyotime, AH158), GFP (Beyotime, AF1483), and GAPDH (Gene Tex, GTX100118), Tubulin (Beyotime, AT819), ACTB (Beyotime, AA128).

Techniques: Activation Assay, Transfection, Western Blot, Control, Immunoprecipitation

Journal: Autophagy

Article Title: Induction of autophagy and suppression of type I IFN secretion by CSFV

doi: 10.1080/15548627.2020.1739445

Figure Lengend Snippet: AKT-MTOR pathway plays an important role in the induction of autophagy by CSFV. (A) PK-15 cells were pretreated with U0126 (7.5 µM), INS (100 ng/mg), compound C (50 µM) or DMSO (control) for 1.5 h, followed by CSFV infection for 1.5 h at an MOI of 1. The host cells were further cultured in fresh medium in the presence of U0126 (7.5 µM), INS (100 ng/mg), compound C (50 µM) or DMSO (control). And then the infected PK-15 cells were studied by electron microscopy at 48 hpi. The average number of autophagosome-like vesicles per cell from at least 20 cells in each group. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). scale bar: 2 µm. Black arrows indicate the structures with the characteristics of autophagosomes. (B) 3D4/2 and PK-15 cells were pretreated with U0126 (7.5 µM), INS (100 ng/mg), compound C (50 µM) or DMSO (control) for 1.5 h, followed by CSFV infection for 1.5 h at an MOI of 1. The host cells were further cultured in fresh medium in the presence of U0126 (7.5 µM), INS (100 ng/mg), compound C (50 µM) or DMSO (control). At 48 hpi, cell samples were analyzed by immunoblotting with antibodies against p-MTOR, LC3B, CSFV-E2, and ACTB (loading control). The level of protein was quantified using Image-Pro Plus. Error bars indicate the mean (± SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA)

Article Snippet: The primary antibodies were specific for p-PRKAA/AMPKα (Thr172) (Cell Signaling Technology, 2535), p-MAPK1/3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology, 9101), MAPK1/3 (ERK1/2; Cell Signaling Technology, 9102), AKT (Cell Signaling Technology, 9272), p-AKT (Ser473; Cell Signaling Technology, 9272), PRKAA/AMPKα (Novus Biologicals, NB100-239), MTOR (Novus Biologicals, NB100-240), BECN1/Beclin 1 (Novus Biologicals, NB110-87,318), CAMKK2/CaMKKβ (Abcam ab168818), LC3B (Novus Biologicals, NB100-2220), p-MTOR (S2448; Abcam, ab109268), HSP90AB1 (Proteintech, 11,405-1-AP), MAVS (Proteintech, 14,341-1-AP), Flag (Beyotime, AF519), HA (Beyotime, AH158), GFP (Beyotime, AF1483), and GAPDH (Gene Tex, GTX100118), Tubulin (Beyotime, AT819), ACTB (Beyotime, AA128).

Techniques: Control, Infection, Cell Culture, Electron Microscopy, Western Blot

Journal: Autophagy

Article Title: Induction of autophagy and suppression of type I IFN secretion by CSFV

doi: 10.1080/15548627.2020.1739445

Figure Lengend Snippet: Proposed model of the signaling network in CSFV-mediated autophagy and CSFV-mediated autophagy suppressing IFN production. CSFV infection induces the suppression of AKT, upregulation of MAPK1/3, and upregulation of PRKAA via [Ca2+]cyto-mediated CAMKK2 activation, leading to the inhibition of MTOR, ultimately leading to the initiation of autophagy. Then CSFV-mediated autophagy suppressed IFN production

Article Snippet: The primary antibodies were specific for p-PRKAA/AMPKα (Thr172) (Cell Signaling Technology, 2535), p-MAPK1/3 (ERK1/2; Thr202/Tyr204; Cell Signaling Technology, 9101), MAPK1/3 (ERK1/2; Cell Signaling Technology, 9102), AKT (Cell Signaling Technology, 9272), p-AKT (Ser473; Cell Signaling Technology, 9272), PRKAA/AMPKα (Novus Biologicals, NB100-239), MTOR (Novus Biologicals, NB100-240), BECN1/Beclin 1 (Novus Biologicals, NB110-87,318), CAMKK2/CaMKKβ (Abcam ab168818), LC3B (Novus Biologicals, NB100-2220), p-MTOR (S2448; Abcam, ab109268), HSP90AB1 (Proteintech, 11,405-1-AP), MAVS (Proteintech, 14,341-1-AP), Flag (Beyotime, AF519), HA (Beyotime, AH158), GFP (Beyotime, AF1483), and GAPDH (Gene Tex, GTX100118), Tubulin (Beyotime, AT819), ACTB (Beyotime, AA128).

Techniques: Infection, Activation Assay, Inhibition