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Image Search Results
Journal: Developmental cell
Article Title: FGF15 Activates Hippo Signaling to Suppress Bile Acid Metabolism and Liver Tumorigenesis.
doi: 10.1016/j.devcel.2018.12.021
Figure Lengend Snippet: Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) Mob1, Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunofluorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-filtration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019
Article Snippet: REAGENT or
Techniques: Western Blot, Microscopy, Phospho-proteomics, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy
Journal: Developmental cell
Article Title: FGF15 Activates Hippo Signaling to Suppress Bile Acid Metabolism and Liver Tumorigenesis.
doi: 10.1016/j.devcel.2018.12.021
Figure Lengend Snippet: Figure 2. FGFR4 and WW45-Mediated Signals Synergistically Modulate Mst1/2 Activities (A) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, WW45, and GAPDH in the lysates of WT or WW45 KO hepatocytes treated for 20 min with indicated FGF19 concentration. (B) Liver-to-body weight ratios of 1-month-old WW45 KO and Mst1/2 DKO mice that were infected with AAV-CTR or AAV-FGF15 at post-natal day 5. (n = 8 mice WW45, Mst1/2 DKO for AAV-CTR; n = 9 mice WW45 for AAV-FGF15; n = 7 mice Mst1/2 DKO for AAV-FGF15). (C) Immunofluorescence microscopy of Yap (red), b-catenin (green), and DAPI (blue) in the liver sections of WT, FGFR4 KO, WW45 KO, and FGFR4 WW45 DKO mice. Scale bars, 20 mm. (D) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, SHP, FGFR4, WW45, and GAPDH in liver tissue lysates from WT, FGFR4 KO, WW45 KO, and FGFR4 WW45 DKO mice. (E) Ki67+ and CK19+ cells by IHC staining in the liver sections from WT, FGFR4 KO, WW45 KO, and FGFR4 WW45 DKO mice. (n = 6). (F and G) A representative liver image (F) and the liver-to-body weight ratios (n = 9 mice per group) (G) of 2-month-old WT, FGFR4 KO, WW45 KO, and FGFR4 WW45 DKO mice. (H and I) A representative liver image (H) and quantification of the size and number of liver tumors (n = 6 mice per group) (I) of 6-month-old WT, FGFR4 KO, WW45 KO, and FGFR4 WW45 DKO mice. (J and K) A representative liver image (J) and the liver-to-body weight ratios (n = 9 mice per group) (K) of 2-month-old WT, FGFR4 WW45 DKO, and FGFR4 WW45 DKO YAP+/ mice. (L) Ki67+ and CK19+ cell quantification by IHC staining in the liver sections from WT, FGFR4 WW45 DKO, and FGFR4 WW45 DKO YAP+/ mice. (n = 6). The data were assessed by Student’s t test and represented as mean ± SD (E, G, K and L) or ± SEM (B and I). ns, not significant (p > 0.05), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared between the indicated groups. See also Figure S2.
Article Snippet: REAGENT or
Techniques: Western Blot, Concentration Assay, Infection, Microscopy, Immunohistochemistry
Journal: Developmental cell
Article Title: FGF15 Activates Hippo Signaling to Suppress Bile Acid Metabolism and Liver Tumorigenesis.
doi: 10.1016/j.devcel.2018.12.021
Figure Lengend Snippet: Figure 3. NF2 Acts as a Molecular Switch of FGFR4 Signaling to Activate Mst1/2 (A) Immunoblot analysis of co-immunoprecipitation of FGFR4 (a-Flag) with NF2 or total liver lysates (input) prepared from WT mice infected with AAV- Flag-FGFR4. (B) FGFR4 and NF2 immune complex in vitro kinase assays. Flag-FGFR4 WT or kinase dead (KD) were immunoprecipitated from transfected 293T cells and incubated with His-NF2 substrate for 30 min at 30C. Immunoblotting of the kinase assay sample as indicated. (C) Immunoblot analysis of co-immunoprecipitation of NF2 (a-Flag) with FGFR4 or total lysates (input) prepared from 293T cells expressing various combinations of HA-tagged FGFR4, Flag-tagged NF2, and treated with or without 100 ng/mL FGF19 for 30 min. (D) Immunoblot analysis of p-tyrosine (p-Tyr) and p-serine/threonine (p-S/T) levels of NF2 in the anti-Flag immunoprecipitates prepared from 293T cells expressing Flag-tagged-NF2 with or without co-transfection of HA-tagged FGFR4. (E) Immunoblot analysis of phospho-NF2 (p-Tyr), Raf1, and NF2 in the anti-NF2 immunoprecipitates or total lysates prepared from WT liver infected with AAV- Flag-FGFR4 or AAV-CTR. (F) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, p-ERK, ERK, NF2, and GAPDH in HepG2 cells expressing control (shCTR) or shNF2 shRNA and treated for indicated times with or without 100 ng/mL FGF19. (G) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, p-ERK, ERK, FGFR4, NF2, and GAPDH in the lysates of 293T cells co-expressing HA-tagged FGFR4 with increasing doses of Flag-tagged NF2.
Article Snippet: REAGENT or
Techniques: Western Blot, Immunoprecipitation, Infection, In Vitro, Transfection, Incubation, Kinase Assay, Expressing, Cotransfection, Control, shRNA
Journal: Developmental cell
Article Title: FGF15 Activates Hippo Signaling to Suppress Bile Acid Metabolism and Liver Tumorigenesis.
doi: 10.1016/j.devcel.2018.12.021
Figure Lengend Snippet: Figure 5. Mst1/2 Phosphorylate and Stabilize SHP (A) Immunoblot analysis of co-immunoprecipitation of SHP (a-Flag) with Mst1, Mst2, and WW45 or total liver lysates (input) prepared from WT mice infected with AAV-FLAG-SHP. (B) His-pull-down assay of His-SHP with GST-Mst2 or GST-WW45. Immunoblotting of pull-down samples and Coomassie blue staining of input samples. (C) Immunoblot analysis of co-immunoprecipitation of SHP with Mst1, Mst2, and WW45 or total liver lysates (input) prepared from WT and WW45 KO hepatocytes. (D) Phos-tag and immunoblot analysis of HA-tagged SHP in the anti-HA immunoprecipitates prepared from 293T cells expressing various combinations of Flag-tagged wild-type Mst2 (WT), kinase-inactive Mst2 (K/R), or WW45. (E) Phos-tag analysis of HA-tagged SHP (WT), SHP (S26A), SHP (T58A), SHP (S28A), or SHP (S26A/S28A/T58A, TripA) co-expressed with Flag-Mst2 and Flag-WW45 in anti-HA immunoprecipitates. (F) Immunoblot analysis of p-SHP (S28), Mst2, Mst2 (K/R), and WW45 in the lysates of 293T cells expressing various combinations of HA-tagged SHP or SHP (S28A), with Flag-tagged Mst2, Mst2(K/R), or WW45. (G) Immunoblot analysis of p-SHP (S28), SHP, p-Mob1, Mob1, and GAPDH in the lysates of hepatocytes treated with or without 3 mM XMU-MP-1 (Mst1/2 inhibitor) for 6 h, 100 ng/mL FGF19 for 20 min or combined. (H) Immunoblot analysis of the ubiquitination of SHP (detected with a-Myc antibody) in the anti-HA immunoprecipitates prepared from 293T cells expressing various combinations of Myc-ubiquitin, HA-SHP, Flag-WW45, and Flag-Mst2.
Article Snippet: REAGENT or
Techniques: Western Blot, Immunoprecipitation, Infection, Pull Down Assay, Staining, Expressing, Ubiquitin Proteomics
Journal: Developmental cell
Article Title: FGF15 Activates Hippo Signaling to Suppress Bile Acid Metabolism and Liver Tumorigenesis.
doi: 10.1016/j.devcel.2018.12.021
Figure Lengend Snippet: Figure 7. Dysregulation of FGFR4-Mst1/2 Signaling in Human HCC Development (A) TBA levels of serum samples of 53 healthy without HCC and 69 patients with HCC. (n = 53 healthy; n = 69 HCC). The data were assessed by Mann-Whitney t test, and represented as mean ± SEM, **p < 0.01 compared between the indicated groups. (B and C) Immunoblot analysis of FGFR4, NF2, p-Yap, p-Mob1, p-ERK, and GAPDH in HCC tissue (T) and adjacent non-tumorous liver tissue (N) isolated from one patient. A total of 6 representative paired samples are shown (B). See Figure S6 for the remaining 54 paired samples. The intensities of the immunoblot bands were quantified using ImageJ software. A heatmap representation of the ratio of the relative expression of the proteins p-Mob1, p-Yap, NF2, FGFR4, or p-ERK in the T and N samples from one patient. Clustering was performed by using Pearson correlation metric and centroid linkage (C). (D) Schematic diagram of the analyses of FRGR4 and NF2 signals in 60 HCC patients. (E) A proposed working model for bile acid metabolism and tumor suppression mediated by the FGF15/19-Hippo signaling. See also Figure S6.
Article Snippet: REAGENT or
Techniques: MANN-WHITNEY, Western Blot, Isolation, Software, Expressing
Journal: Nature communications
Article Title: Structural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3 signaling.
doi: 10.1038/s41467-023-38869-9
Figure Lengend Snippet: Fig. 4 | PEAK3:14-3-3 forms a stable high affinity heterocomplex. a Recombinant PEAK3FL from insect cells elutes on size exclusion chromatography (SEC; S200 10/ 300) as a high affinity stoichiometric complex of dimeric PEAK3 with a 14-3-3ε,ζ heterodimer, results supported by SDS-PAGE analysis of eluted fractions (n = 3, independent experiments) (see also Supplementary Fig. 4a, source data). b Immu- noprecipitation of PEAK3 WT and PEAK3 S69A showing S69 is required for 14-3-3 co-immunoprecipitation from cells (n = 3 biologically independent samples, see
Article Snippet: Nature Communications | (2023) 14:3542 13 Recombinant kinase domains for in vitro phosphorylation reactions Constructs for expression of
Techniques: Recombinant, Size-exclusion Chromatography, SDS Page, Immunoprecipitation
Journal: Cell reports. Medicine
Article Title: Targeting nucleic acid sensors in tumor cells to reprogram biogenesis and RNA cargo of extracellular vesicles for T cell-mediated cancer immunotherapy.
doi: 10.1016/j.xcrm.2023.101171
Figure Lengend Snippet: Figure 1. Tumor cell-intrinsic RIG-I signaling mediates the release of immunogenic EVs B16.OVA melanoma cells were transfected with a RIG-I ligand (3p-RNA, RIG-I-EVs), interferon-stimulating DNA (cGAS-EVs), or synthetic RNA (synRNA-EVs). Extracellular vesicles (EVs) were enriched from the culture supernatant of treated and untreated (ctrl-EVs) cells. Precipitation of 3pRNA-liposomes in the absence of tumor cells was performed as negative control (mock-EVs). (A) IFN-b release (ELISA) by dendritic cells (DCs) exposed to tumor cell-derived EV samples. Some DCs were directly transfected with in vitro transcribed 3pRNA as positive control. (B–D) IFN-b release from DCs exposed to RIG-I-EV preparations: (B) prepared from different melanoma clones that lack specific downstream signaling com- ponents of nucleic acid receptor pathways; (C) enriched from various murine tumor cell lines: mammary (4T1), pancreatic (Panc02), colon (C26) carcinoma; and (D) enriched by precipitation or size-exclusion chromatography. All data are presented as mean values ± SEM of at least quadruplicate technical replicates per group and are representative of two independent experiments. Asterisks without brackets indicate statistical comparison with Ctrl-EV-treated cells. (E) Presence of the antigen OVA (by western blot) in melanoma cell EV samples. (F) Treatment model. C57BL6/j mice were repeatedly injected subcutaneously with tumor EV samples prepared from cultures of wild-type, RIG-I-deficient (RIG-I/), or IRF3/7-deficient (IRF3/7/) B16.OVA melanoma cells. (G) IFN-g release by CD8+ T cells from draining lymph nodes after ex vivo OVA restimulation (flow cytometry). Data are presented as mean values ± SEM of n = 4–5 individual mice per group and were pooled from two independent experiments. Unstim, unstimulated. See also Figures S1–S3.
Article Snippet: REAGENT or
Techniques: Transfection, Liposomes, Negative Control, Enzyme-linked Immunosorbent Assay, Derivative Assay, In Vitro, Positive Control, Clone Assay, Size-exclusion Chromatography, Comparison, Western Blot, Injection, Ex Vivo, Cytometry