mouse monoclonal anti myh3 (Developmental Studies Hybridoma Bank)

Developmental Studies Hybridoma Bank mouse monoclonal anti myh3

Mouse Monoclonal Anti Myh3, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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bpmonwin software version 1.35 (IITC Life Science)

IITC Life Science bpmonwin software version 1.35

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pdquest software (Bio-Rad)

Bio-Rad pdquest software

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cycle ergometer velotron dynafit pro (Velotron Inc)

Velotron Inc cycle ergometer velotron dynafit pro

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resource source identifier antibodies phospho mob1 thr35 (Cell Signaling Technology Inc)

Cell Signaling Technology Inc resource source identifier antibodies phospho mob1 thr35
$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) <t>Mob1,</t> Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$
Resource Source Identifier Antibodies Phospho Mob1 Thr35, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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topkapi software (Abbott Laboratories)

Abbott Laboratories topkapi software
$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) <t>Mob1,</t> Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$
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ms search software (National Institute of Standards and Technology)

National Institute of Standards and Technology ms search software
$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) <t>Mob1,</t> Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$
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stereoinvestigator (v. 4.35) software program (MBF Bioscience)

MBF Bioscience stereoinvestigator (v. 4.35) software program
$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) <t>Mob1,</t> Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$
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molcharge (OpenEye Scientific Software Inc)

OpenEye Scientific Software Inc molcharge
$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) <t>Mob1,</t> Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$
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calphad software thermo-calc (Thermo Fisher)

Thermo Fisher calphad software thermo-calc
$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) <t>Mob1,</t> Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$
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software version 9.4 (SAS institute)

SAS institute software version 9.4
$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) <t>Mob1,</t> Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$
Software Version 9.4, supplied by SAS institute, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dragonfly 2020.1 software (Object Research Systems (ORS) Inc)

Object Research Systems (ORS) Inc dragonfly 2020.1 software
$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) <t>Mob1,</t> Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$
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Image Search Results

Journal: iScience

Article Title: Uhrf1 governs the proliferation and differentiation of muscle satellite cells

doi: 10.1016/j.isci.2022.103928

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Myh3 , DSHB , cat# F1.652; RRID: AB_528358.

Techniques: Recombinant, Western Blot, Imaging, Multiplex Assay, Library Quantification, Methylation, Electrophoresis, Software

$Figure 1. FGF15/19 Activates Mst1/2 through Hepatic Receptor FGFR4 (A) Diagram of the parabiosis model where two mice were surgically joined. Immunoblot analysis of phosphorylated (p-) Mob1, Mob1, p-Yap, Yap, Mst1, Mst2, and GAPDH in the liver lysates of the indicated parabionts. (B) Immunoblot analysis of p-Mob1, Mob1, p-Yap, Yap, and GAPDH in the lysates of primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse normal or heat-denatured serum for indicated times. (C) Immunoﬂuorescence microscopy of Yap (red), a-tubulin (green), and DAPI (for nuclear counterstain, blue) in primary hepatocytes treated with 15% WT or Mst1/2 DKO mouse serum for 20 min. Scale bars, 10 mm. (D) Schematic diagram of the experiments conducted to determine the potential Mst1/2-activating components in Mst1/2 DKO mouse serum. The assay was performed with Mst1/2 DKO mouse serum using size-exclusion gel-ﬁltration FPLC to separate the serum proteins according to their size, and the different fractions were analyzed for their ability to induce Mob1 phosphorylation in hepatocytes. The active and inactive fractions were analyzed by liquid chromatog- raphy-tandem mass spectrometry (LC-MS/MS) to identify proteins that were more abundant in the active fractions than in the inactive fractions. (legend continued on next page) 2 Developmental Cell 48, 1–15, February 25, 2019$

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 RESOURCE SOURCE IDENTIFIER Antibodies Phospho-MOB1 (Thr35) (D2F10) Rabbit mAb Cell Signaling Technology Cat#8699; RRID:AB_11139998 MOB1 (E1N9D) Rabbit Cell Signaling Technology Cat#13730 Phospho-Yap (S127) (rabbit) Cell Signaling Technology Cat#4911S; RRID: AB_2218913 Yap (rabbit) Cell Signaling Technology Cat#4912S; RRID:AB_10694682 GAPDH [D16H11] (rabbit) Cell Signaling Technology Cat#5174; RRID:AB_10622025 a-Tubulin [11H10] (rabbit) Cell Signaling Technology Cat#2125; RRID:AB_2619646 Merlin (rabbit) Cell Signaling Technology Cat#6995; RRID:AB_10828709 p44/42 MAPK (Erk1/2) (3A7) Mouse Cell Signaling Technology Cat#9107; RRID:AB_10695739 HA-Tag [C29F4] (rabbit) Cell Signaling Technology Cat#3724; RRID:AB_1549585 HA-Tag (rabbit) Proteintech Cat#51064-2-AP RRID: AB_11042321 Flag-Tag (rabbit) Proteintech Cat#20543-1-AP RRID:AB_11232216 Myc-Tag (rabbit) Proteintech Cat# 16286-1-AP RRID:AB_11182162 phospho-Erk1/2 (rabbit) Cell Signaling Technology Cat#4370; RRID:AB_2315112 Myc-Tag (rabbit) Cell Signaling Technology Cat#2278; RRID:AB_10693332 Phospho-Tyrosine (Mouse) Cell Signaling Technology Cat#9411; RRID:AB_331228 Mst1(rabbit) Cell Signaling Technology Cat#3682; RRID: AB_2144632 Mst2 (rabbit) Cell Signaling Technology Cat#3952; RRID: AB_2196471 Lats1 [C66B5] (rabbit) Cell Signaling Technology Cat#3477; RRID: AB_2133513 SAV1 [M02] (mouse) Abnova Cat#H00060485-M02 RRID: AB_547968 FGFR4 (rabbit) Abcam Cat#ab178396; RRID:N/A b-catenin [15B8] (mouse) Abcam Cat#ab6301; RRID:AB_305406 Phospho-Ser/Thr antibody (rabbit) Abcam Cat#ab17464; RRID:AB_443891 Glutamine synthase (rabbit) Abcam Cat#ab49873; RRID:AB_880241 Cyp7a1 (mouse) Millipore Cat#MABD42 Brdu [BU-1] (mouse) Thermo Fisher Cat#MA3-071; RRID:AB_10986341 Flag-Tag (mouse) Sigma-Aldrich Cat#F1804; RRID: AB_262044 6X His-Tag antibody (mouse) Abcam Cat#ab18184; RRID:AB_444306 GST-Tag (mouse) Proteintech Cat#66001-2-Ig RRID:N/A Ki67 Cell Signaling Technology Cat#12202; RRID:AB_2620142 CK19 DSHB AB_2133570; RRID:AB_2133570 SHP (Rabbit) This paper N/A Phospho-SHP (S28) (Rabbit) This paper N/A (Continued on next page) e1 Developmental Cell 48, 1–15.e1–e9, February 25, 2019

Techniques: Western Blot, Microscopy, Phospho-proteomics, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

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.

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.

Techniques: Western Blot, Concentration Assay, Infection, Microscopy, Immunohistochemistry

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.

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.

Techniques: Western Blot, Immunoprecipitation, Infection, In Vitro, Transfection, Incubation, Kinase Assay, Expressing, Cotransfection, Control, shRNA

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.

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.

Techniques: Western Blot, Immunoprecipitation, Infection, Pull Down Assay, Staining, Expressing, Ubiquitin Proteomics

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.

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.

Techniques: MANN-WHITNEY, Western Blot, Isolation, Software, Expressing

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