Journal: Molecular and Cellular Biology

Article Title: In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts

doi: 10.1128/mcb.25.1.66-77.2005

Figure Lengend Snippet: FIG. 2. Signaling in mutant mice in response to GH injections. (A) Predicted binding of signaling and adaptor molecules to the cytoplasmic domain of the GHR of the WT, mutant 569, and mutant 391. (B to D) Livers from the bGH- and saline-injected 19-day-old mice were used in immunoprecipitation analyses 15 min after the injections. Loading for each of the proteins was confirmed by using antibodies specific for an appropriate protein. (B) Antiphosphotyrosine (PY) immunoblot of JAK2 immunoprecipitated from liver homogenate. (C) Immunoblot for active ERK1/2 (Phospho-p44/42 mitogen-activated protein kinase) from liver homogenate. (D) Antiphosphotyrosine (PY) immunoblot of STAT5 immunoprecipitated from liver homogenate. (E to G) Densitometric quantification of the blots from panels B to D. Signals for the activated JAK2 (E), ERK1/2 (F), and STAT5a/b (G) were normalized for loading. The data for all graphs are presented as means the SEM.

Article Snippet: Liver homogenates were used in immunoprecipitation with JAK2 (sc-278) and STAT5 (sc-835) antibodies from Santa Cruz Biotech (Santa Cruz, Calif.), and samples were separated on a polyacrylamide gel, blotted onto a nitrocellulose membrane, and probed with antiphosphotyrosine 4G10 antibody.

Techniques: Mutagenesis, Binding Assay, Saline, Injection, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 1. Tyrosine phosphorylation of Jaks and Stat proteins by activated EphA4. A, COS7 cells were transfected with vector (V), EphA4 (A4), or kinase- dead (KD) mutant of EphA4, and the cell lysate was immunoblotted by anti-phos- pho-Stat1 and anti-phospho-Stat3 anti- bodies. Tyrosine phosphorylation of Stat1 (left panel) and Stat3 (right panel) was induced in COS7 cells that overexpressed EphA4 but not the kinase-dead mutant (upper panels). The membrane was stripped and re-probed with antibody against Stat3 or the -isoform of Stat1 to indicate similar loading (middle panels). Both the wild-type and kinase-dead mu- tant was expressed at comparable level, as illustrated by Western blot with anti- EphA4 antibody (lower panels). B, induc- tion of Stat1 transcriptional activity by EphA4. COS7 cells were transfected with vector (V), kinase-dead (KD) mutant of EphA4 or EphA4 (A4), together with a luciferase reporter construct that was linked to Stat1-responsive enhancer ele- ment (GAS-Luc) or control construct that lacked the enhancer (pTA-Luc). The level of luminescence, which corresponded to the activity of luciferase, was measured (mean S.E., n 3). C, Western blot using antibodies against phospho-Jak2 and phospho-Tyk2 (left panel) showed that tyrosine phosphorylation of Jak2 and Tyk2 was induced in COS7 cells that overexpressed EphA4 but not EphA4KD. Similar loading was indicated by re-prob- ing the membrane with anti-Jak2 anti- body. The induction of Jak2 phosphoryla- tion by EphA4 was further verified by immunoprecipitating (IP) Jak2 from the cell lysate of transfected COS7 cells and immunoblotted with anti-phosphotyrosine antibody (right panel). Similar loading was indicated by re-probing the mem- brane with anti-Jak2 antibody.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Mutagenesis, Membrane, Western Blot, Activity Assay, Luciferase, Construct, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 2. Activated EphA4 induced ty- rosine phosphorylation of Stat1 and Stat3 via Jak2. A, COS7 cells were transfected with EphA4 (A4) or kinase- dead (KD) mutant of EphA4. Two days after transfection, the cells were treated with Me2SO or the Jak2 inhibitor AG490 (100 M) for 6 h before cell lysate was collected and immunoblotted with anti- bodies against phospho-Stat1, phospho- Stat3 (left panel), phospho-Jak2, and phospho-Tyk2 (right panel). The presence of AG490 significantly reduced the induc- tion of Jak/Stat tyrosine phosphorylation by EphA4. Immunoblotting the mem- branes with antibodies specific for Stat1, Stat3, and Jak2 indicated similar loading. V, vector. B, COS7 cells were transfected with the kinase-dead (KD) mutant of EphA4 or EphA4 (A4) and treated with Me2SO or different concentrations of AG490 (50–100 M) for 6 h. The lysate was immunoblotted by antibodies against phospho-Stat3 and phospho-FAK (left panel). AG490 inhibited the tyrosine phosphorylation of Stat3 and FAK in- duced by overexpressed EphA4. EphA4 or EphA4KD was immunoprecipitated from transfected cells by anti-EphA4 antibody and immunoblotted by anti-phospho-tyro- sine antibody (right panel). The autophos- phorylation of EphA4 was not affected by AG490 even at higher concentration (100 M). Immunoblotting the membrane with anti-EphA4 indicated that similar amount of EphA4 was immunoprecipitated in each sample.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Immunoprecipitation, Concentration Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 1. Tyrosine phosphorylation of Jaks and Stat proteins by activated EphA4. A, COS7 cells were transfected with vector (V), EphA4 (A4), or kinase- dead (KD) mutant of EphA4, and the cell lysate was immunoblotted by anti-phos- pho-Stat1 and anti-phospho-Stat3 anti- bodies. Tyrosine phosphorylation of Stat1 (left panel) and Stat3 (right panel) was induced in COS7 cells that overexpressed EphA4 but not the kinase-dead mutant (upper panels). The membrane was stripped and re-probed with antibody against Stat3 or the -isoform of Stat1 to indicate similar loading (middle panels). Both the wild-type and kinase-dead mu- tant was expressed at comparable level, as illustrated by Western blot with anti- EphA4 antibody (lower panels). B, induc- tion of Stat1 transcriptional activity by EphA4. COS7 cells were transfected with vector (V), kinase-dead (KD) mutant of EphA4 or EphA4 (A4), together with a luciferase reporter construct that was linked to Stat1-responsive enhancer ele- ment (GAS-Luc) or control construct that lacked the enhancer (pTA-Luc). The level of luminescence, which corresponded to the activity of luciferase, was measured (mean S.E., n 3). C, Western blot using antibodies against phospho-Jak2 and phospho-Tyk2 (left panel) showed that tyrosine phosphorylation of Jak2 and Tyk2 was induced in COS7 cells that overexpressed EphA4 but not EphA4KD. Similar loading was indicated by re-prob- ing the membrane with anti-Jak2 anti- body. The induction of Jak2 phosphoryla- tion by EphA4 was further verified by immunoprecipitating (IP) Jak2 from the cell lysate of transfected COS7 cells and immunoblotted with anti-phosphotyrosine antibody (right panel). Similar loading was indicated by re-probing the mem- brane with anti-Jak2 antibody.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Mutagenesis, Membrane, Western Blot, Activity Assay, Luciferase, Construct, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 2. Activated EphA4 induced ty- rosine phosphorylation of Stat1 and Stat3 via Jak2. A, COS7 cells were transfected with EphA4 (A4) or kinase- dead (KD) mutant of EphA4. Two days after transfection, the cells were treated with Me2SO or the Jak2 inhibitor AG490 (100 M) for 6 h before cell lysate was collected and immunoblotted with anti- bodies against phospho-Stat1, phospho- Stat3 (left panel), phospho-Jak2, and phospho-Tyk2 (right panel). The presence of AG490 significantly reduced the induc- tion of Jak/Stat tyrosine phosphorylation by EphA4. Immunoblotting the mem- branes with antibodies specific for Stat1, Stat3, and Jak2 indicated similar loading. V, vector. B, COS7 cells were transfected with the kinase-dead (KD) mutant of EphA4 or EphA4 (A4) and treated with Me2SO or different concentrations of AG490 (50–100 M) for 6 h. The lysate was immunoblotted by antibodies against phospho-Stat3 and phospho-FAK (left panel). AG490 inhibited the tyrosine phosphorylation of Stat3 and FAK in- duced by overexpressed EphA4. EphA4 or EphA4KD was immunoprecipitated from transfected cells by anti-EphA4 antibody and immunoblotted by anti-phospho-tyro- sine antibody (right panel). The autophos- phorylation of EphA4 was not affected by AG490 even at higher concentration (100 M). Immunoblotting the membrane with anti-EphA4 indicated that similar amount of EphA4 was immunoprecipitated in each sample.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Immunoprecipitation, Concentration Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 3. EphA4 interacted with Jak2 in vitro and in vivo. A, EphA4 (A4), kinase-dead (KD) mutant of EphA4, or vector (V) was transfected into COS7 cells, and the cell lysate was immunoprecipitated (IP) by anti-Jak2 antibody and immunoblotted by antibody against EphA4 (upper panel). In the negative control, rabbit IgG was used in the immunoprecipitation. Both the wild-type EphA4 and the kinase-dead mutant was co-immunoprecipitated with Jak2. The association between EphA4 and Jak2 was verified in reciprocal immunoprecipitation, in which Jak2 was co-immunoprecipitated by anti-EphA4 antibody (middle panel). The association between Jak2 and EphA4 was specific, since Stat1 was not immunoprecipitated (IP) by anti-EphA4 antibody (lower panel). Similar experiments showed that Stat3 was not co-immunoprecipitated with EphA4 (data not shown). B, the N-terminal (JH domains 3–7) or C-terminal half (JH domains 1–3) of Jak2 was linked to GST, and the fusion proteins were purified and used to pull down EphA4 from lysate of transfected COS7 cells. EphA4 was mainly pulled down by the N-terminal half of Jak2 that contained the JH3 to JH7 domains (upper panel). The specificity of the pull-down assay was indicated by the absence of EphA4 in the pull-down reaction using GST alone or GST-CBD2 (cytokine-binding domain 2) of LIF receptor. Similar amounts of different GST fusion proteins were used in the pull-down assay, as confirmed by re-blotting the membrane with anti-GST antibody (lower panel). C, Jak2 was associated with EphA4 in neurons. Cortical neurons, which expressed endogenous EphA4 and Jak2, were cultured for 15 days, and the cell lysate was subjected to immunoprecipitation by anti-Jak2 antibody. EphA4 was detected in the immunoprecipitated reaction by anti-Jak2 antibody but not rabbit IgG (left panel). Likewise, Jak2 was reciprocally immunoprecipitated by anti-EphA4 antibody but not rabbit IgG (right panel). D, the interaction between EphA4 and Jak2 occurred in vivo. Membrane proteins of rat brain (Br) at different developmental stages (embryonic day 20, postnatal day 14, and adult) were subjected to immunoprecipitation by anti-Jak2 antibody, and the product was immunoblotted by EphA4 antibody. Significantly stronger signal of EphA4 was detected in the immunoprecipitation reaction by anti-Jak2 antibody compared with the IgG negative control (upper panel). The association between EphA4 and Jak2 in postnatal day 14 rat brain was verified by the reciprocal immunoprecipitation (lower panel, left). EphA4 was also co-immunoprecipitated by anti-Jak2 antibody from rat hindlimb muscle (Mus) at embryonic day 20 (lower panel, right).

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: In Vitro, In Vivo, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Negative Control, Purification, Pull Down Assay, Binding Assay, Membrane, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 4. Induction of Stat3 tyrosine phosphorylation by ephrin-A1 in cultured myotubes. A, the muscle cell line C2C12, which expressed endogenous EphA4, was differentiated into myotubes and treated with ephrin-A1 (2 g/ml) for 2–20 min. Equivalent amount of Fc fragment of IgG (Fc) was used as the negative control. The cell lysate was immunoblotted by anti-phospho-Stat3 antibody (upper panel). Induction of Stat3 tyrosine phosphorylation was observed upon treatment with ephrin-A1 compared with the Fc negative control. The membrane was stripped and re-probed with Stat3 to indicate similar loading (lower panel). B, the induction of Stat3 tyrosine phosphorylation by ephrin-A1 was dependent on Jak2. Differentiated C2C12 myotubes were pre-treated with AG490 (75 or 100 M) or Me2SO (DMSO) for 6 h before treating with ephrin-A1 or Fc in the presence of AG490 or Me2SO. In the negative control (Con), neither Fc nor ephrin-A1 was added. The cell lysate was immunoblotted by anti-phospho-Stat3 antibody (upper panel). The ephrin-A1-induced tyrosine phosphorylation of Stat3 was observed in the presence of Me2SO but not the Jak2 inhibitor AG490. Similar loading was indicated by blotting the membrane with Stat3. The specificity of the action of AG490 was confirmed by immunoblotting the lysate with anti-phospho-ERK antibody (lower panel). The threonine/tyrosine phosphorylation of ERK-1/2 was reduced after treatment with ephrin-A1 for 10 min in the presence of Me2SO, and AG490 did not affect the ephrin-A1-induced down-regulation of ERK-1/2 phosphorylation.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Phospho-proteomics, Cell Culture, Negative Control, Membrane, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 5. Localization of Jak/Stat proteins at the neuromuscular junction. Double immunofluorescence staining of adult rat gastrocne- mius muscle sections indicated the co-localization of Jak2, Stat1, and Stat3 (left panel) with acetylcholine receptor (AChR), which was de- tected by rhodamine-conjugated -bungarotoxin (right panel). The spec- ificity of staining was confirmed by the absence of fluorescence when the antibodies against Jak2 and Stat1 were pre-incubated with the corresponding peptides against which the antibodies were raised or in the negative control in which the primary antibody was substituted by rabbit IgG.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Double Immunofluorescence Staining, Staining, Fluorescence, Incubation, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 6. Regulation of acetylcholinesterase expression by ephrin/Eph signaling in muscle was dependent on Jak2. A, differentiated C2C12 myotubes were treated with Me2SO (DMSO) or AG490 (75 M) for 24 h, and total RNA was analyzed in Northern blot and hybridized with acetylcholinesterase (AChE, upper panel). The level of AChE transcript was significantly reduced by AG490. Similar results were obtained when lower concentration (25 M) of AG490 was added (data not shown). The membrane was re-probed with tropomyosin to indicate similar loading of RNA (lower panel). The positions of ribosomal RNAs (28 S and 18 S) were indicated. B, differentiated C2C12 myotubes were treated with ephrin-A1 or Fc (2 g/ml) for 24 h in the presence of Me2SO or AG490 (75 M). Northern blot analysis with AChE (upper panel) and fibronectin (lower panel) showed that ephrin-A1 decreased the expression of fibronectin transcript, and the reduction was not affected by the presence of AG490. C, differentiated C2C12 myotubes were pre-treated with Me2SO or AG490 (75 M) for 3 h and then incubated with ephrin-A1 or Fc (2 g/ml) for 24 h in the presence of Me2SO or AG490. The cell lysate was immunoblotted by antibody against AChE (upper panel). The expression of AChE protein was increased by ephrin-A1, and the induction was abolished by AG490. The membrane was stripped and re-probed with antibody against ERK-1/2 to indicate similar loading (lower panel). The intensity of the AChE bands was quantified after normalization with that of the ERK-2 bands (mean S.E., n 4). The difference in band intensity between Fc and ephrin-A1 in the presence of Me2SO was statistically significant (*, p 0.001). D, expression of AChE protein in muscle of wild-type and EphA4 knockout mice. Cytosolic and membrane fractions of hindlimb muscles from wild-type (/) and EphA4 null mice (/) at postnatal day 14 were immunoblotted by antibody against AChE (upper panel). The level of AChE protein was significantly reduced in EphA4/ mice, and the difference was consistently observed in four different pairs of wild-type and homozygous null mice. The membrane was stripped and re-probed with antibody against Stat3 (lower panel) to indicate similar loading of proteins between the / and / mice.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Expressing, Northern Blot, Concentration Assay, Membrane, Incubation, Knock-Out, Muscles

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 1. Tyrosine phosphorylation of Jaks and Stat proteins by activated EphA4. A, COS7 cells were transfected with vector (V), EphA4 (A4), or kinase- dead (KD) mutant of EphA4, and the cell lysate was immunoblotted by anti-phos- pho-Stat1 and anti-phospho-Stat3 anti- bodies. Tyrosine phosphorylation of Stat1 (left panel) and Stat3 (right panel) was induced in COS7 cells that overexpressed EphA4 but not the kinase-dead mutant (upper panels). The membrane was stripped and re-probed with antibody against Stat3 or the -isoform of Stat1 to indicate similar loading (middle panels). Both the wild-type and kinase-dead mu- tant was expressed at comparable level, as illustrated by Western blot with anti- EphA4 antibody (lower panels). B, induc- tion of Stat1 transcriptional activity by EphA4. COS7 cells were transfected with vector (V), kinase-dead (KD) mutant of EphA4 or EphA4 (A4), together with a luciferase reporter construct that was linked to Stat1-responsive enhancer ele- ment (GAS-Luc) or control construct that lacked the enhancer (pTA-Luc). The level of luminescence, which corresponded to the activity of luciferase, was measured (mean S.E., n 3). C, Western blot using antibodies against phospho-Jak2 and phospho-Tyk2 (left panel) showed that tyrosine phosphorylation of Jak2 and Tyk2 was induced in COS7 cells that overexpressed EphA4 but not EphA4KD. Similar loading was indicated by re-prob- ing the membrane with anti-Jak2 anti- body. The induction of Jak2 phosphoryla- tion by EphA4 was further verified by immunoprecipitating (IP) Jak2 from the cell lysate of transfected COS7 cells and immunoblotted with anti-phosphotyrosine antibody (right panel). Similar loading was indicated by re-probing the mem- brane with anti-Jak2 antibody.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Mutagenesis, Membrane, Western Blot, Activity Assay, Luciferase, Construct, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 2. Activated EphA4 induced ty- rosine phosphorylation of Stat1 and Stat3 via Jak2. A, COS7 cells were transfected with EphA4 (A4) or kinase- dead (KD) mutant of EphA4. Two days after transfection, the cells were treated with Me2SO or the Jak2 inhibitor AG490 (100 M) for 6 h before cell lysate was collected and immunoblotted with anti- bodies against phospho-Stat1, phospho- Stat3 (left panel), phospho-Jak2, and phospho-Tyk2 (right panel). The presence of AG490 significantly reduced the induc- tion of Jak/Stat tyrosine phosphorylation by EphA4. Immunoblotting the mem- branes with antibodies specific for Stat1, Stat3, and Jak2 indicated similar loading. V, vector. B, COS7 cells were transfected with the kinase-dead (KD) mutant of EphA4 or EphA4 (A4) and treated with Me2SO or different concentrations of AG490 (50–100 M) for 6 h. The lysate was immunoblotted by antibodies against phospho-Stat3 and phospho-FAK (left panel). AG490 inhibited the tyrosine phosphorylation of Stat3 and FAK in- duced by overexpressed EphA4. EphA4 or EphA4KD was immunoprecipitated from transfected cells by anti-EphA4 antibody and immunoblotted by anti-phospho-tyro- sine antibody (right panel). The autophos- phorylation of EphA4 was not affected by AG490 even at higher concentration (100 M). Immunoblotting the membrane with anti-EphA4 indicated that similar amount of EphA4 was immunoprecipitated in each sample.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Immunoprecipitation, Concentration Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 3. EphA4 interacted with Jak2 in vitro and in vivo. A, EphA4 (A4), kinase-dead (KD) mutant of EphA4, or vector (V) was transfected into COS7 cells, and the cell lysate was immunoprecipitated (IP) by anti-Jak2 antibody and immunoblotted by antibody against EphA4 (upper panel). In the negative control, rabbit IgG was used in the immunoprecipitation. Both the wild-type EphA4 and the kinase-dead mutant was co-immunoprecipitated with Jak2. The association between EphA4 and Jak2 was verified in reciprocal immunoprecipitation, in which Jak2 was co-immunoprecipitated by anti-EphA4 antibody (middle panel). The association between Jak2 and EphA4 was specific, since Stat1 was not immunoprecipitated (IP) by anti-EphA4 antibody (lower panel). Similar experiments showed that Stat3 was not co-immunoprecipitated with EphA4 (data not shown). B, the N-terminal (JH domains 3–7) or C-terminal half (JH domains 1–3) of Jak2 was linked to GST, and the fusion proteins were purified and used to pull down EphA4 from lysate of transfected COS7 cells. EphA4 was mainly pulled down by the N-terminal half of Jak2 that contained the JH3 to JH7 domains (upper panel). The specificity of the pull-down assay was indicated by the absence of EphA4 in the pull-down reaction using GST alone or GST-CBD2 (cytokine-binding domain 2) of LIF receptor. Similar amounts of different GST fusion proteins were used in the pull-down assay, as confirmed by re-blotting the membrane with anti-GST antibody (lower panel). C, Jak2 was associated with EphA4 in neurons. Cortical neurons, which expressed endogenous EphA4 and Jak2, were cultured for 15 days, and the cell lysate was subjected to immunoprecipitation by anti-Jak2 antibody. EphA4 was detected in the immunoprecipitated reaction by anti-Jak2 antibody but not rabbit IgG (left panel). Likewise, Jak2 was reciprocally immunoprecipitated by anti-EphA4 antibody but not rabbit IgG (right panel). D, the interaction between EphA4 and Jak2 occurred in vivo. Membrane proteins of rat brain (Br) at different developmental stages (embryonic day 20, postnatal day 14, and adult) were subjected to immunoprecipitation by anti-Jak2 antibody, and the product was immunoblotted by EphA4 antibody. Significantly stronger signal of EphA4 was detected in the immunoprecipitation reaction by anti-Jak2 antibody compared with the IgG negative control (upper panel). The association between EphA4 and Jak2 in postnatal day 14 rat brain was verified by the reciprocal immunoprecipitation (lower panel, left). EphA4 was also co-immunoprecipitated by anti-Jak2 antibody from rat hindlimb muscle (Mus) at embryonic day 20 (lower panel, right).

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: In Vitro, In Vivo, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Negative Control, Purification, Pull Down Assay, Binding Assay, Membrane, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Identification of the Jak/Stat Proteins as Novel Downstream Targets of EphA4 Signaling in Muscle

doi: 10.1074/jbc.m313356200

Figure Lengend Snippet: FIG. 5. Localization of Jak/Stat proteins at the neuromuscular junction. Double immunofluorescence staining of adult rat gastrocne- mius muscle sections indicated the co-localization of Jak2, Stat1, and Stat3 (left panel) with acetylcholine receptor (AChR), which was de- tected by rhodamine-conjugated -bungarotoxin (right panel). The spec- ificity of staining was confirmed by the absence of fluorescence when the antibodies against Jak2 and Stat1 were pre-incubated with the corresponding peptides against which the antibodies were raised or in the negative control in which the primary antibody was substituted by rabbit IgG.

Article Snippet: Antibodies and Reagents—The antibodies against tyrosine-phosphorylated Stat1, Stat3, Tyk2, FAK, and threonine/tyrosine-phosphorylated ERK-1/2 were purchased from Cell Signaling Inc., and the antibody against tyrosine-phosphorylated Jak2 was obtained from Upstate Biotechnology, Inc. Rabbit polyclonal antibodies that recognized Stat1 (sc-346), Stat3 (sc-7179), Jak2 (sc-278), and EphA4 (sc-921) were purchased from Santa Cruz Biotechnology, and the monoclonal antibodies that recognized AChE and the -isoform of Stat1 were purchased from Transduction Laboratories and Zymed Laboratories Inc., respectively.

Techniques: Double Immunofluorescence Staining, Staining, Fluorescence, Incubation, Negative Control