Journal: The Journal of Cell Biology

Article Title: α-Syntrophin regulates ARMS localization at the neuromuscular junction and enhances EphA4 signaling in an ARMS-dependent manner

doi: 10.1083/jcb.200412008

Figure Lengend Snippet: ARMS and α-syntrophin enhanced the EphA4-induced Jak/Stat activation. (A) COS7 cells were transfected with EphA4, EphA4 KD mutant, or ARMS, and cell lysate was immunoblotted with antibodies against phosphorylated Tyr 1007/1008 of mouse Jak2, Tyr 1054/1055 of human Tyk2, Tyr 701 of Stat1, and Thr 202 /Tyr 204 of ERK1/2. p-Tyr; phosphorylated Tyr. (B) Quantification of the Western blots. y axis represents the fold change of tyrosine phosphorylation. n = 3; *, P < 0.05; **, P < 0.005. (C) COS7 cells were transfected with EphA4, ARMS, α-syntrophin, or α-syntrophin mPDZ, and cell lysate was immunoblotted with antibodies against phosphorylated Tyr 1007/1008 of mouse Jak2, Tyr 1054/1055 of human Tyk2, Tyr 701 of Stat1, and Thr 202 /Tyr 204 of ERK1/2. (D) Quantification of the Western blots. n = 3; *, P < 0.05; **, P < 0.005. Error bars represent the SEM.

Article Snippet: Polyclonal anti-HA antibodies, EphA4 antibody (sc-921), and Jak2 antibody (sc-278) were purchased from Santa Cruz Biotechnology, Inc., and the mAb that recognizes the α-isoform of Stat1 was obtained from Zymed Laboratories.

Techniques: Activation Assay, Transfection, Mutagenesis, Western Blot, Phospho-proteomics

Journal: Frontiers in Physiology

Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus

doi: 10.3389/fphys.2022.956116

Figure Lengend Snippet: Effects of acute exercise on metabolic parameters and TUB regulation. (A) Body weight (g); (B) 24 h of food intake (g); (C) serum IL-6 levels (pg/ml); and (D) Western blot from hypothalamic lysates showing IL-6 expression in C57BL/6J mice (12–15 weeks of age) under resting conditions (Sed) or after swimming exercise (Exe). B6- tub/+ or B6- tub/tub mice (6–8 weeks of age) were divided into the following groups: B6- tub/+ : sedentary plus vehicle; sedentary plus IL-6 ICV; exercise plus vehicle; exercise plus ABIL6 (IL-6 antibody); B6- tub/tub : exercise plus vehicle. (E) Evaluation of the cumulative food intake (g) at 4 h, 12 h, and 24 h; (H) AgRP and (I) POMC mRNA expression in the arcuate nucleus of the hypothalamus. Western blot showing (F) TUB tyrosine phosphorylation and (G) TUB associated with JAK2 from hypothalamus lysates. The exercise was swimming. All mice were male. ICV: intracerebroventricular. All values are expressed as means ± standard deviation (SD). (A–C) : Sed ( n = 7) and Exe ( n = 6). (D,F,G) : ( n = 4 each group). (E,H,I) : ( n = 5 each group). Unpaired two-tailed t -tests were used to analyze (A–D) ; two-way ANOVA was used to analyze (E) ; one-way ANOVA with Tukey’s multiple comparisons tests was used to analyze (F–I) . * p < 0.0001 vs. other groups; & p < 0.05 vs. other groups; # [Sed + IL-6 vs. Sed + Veh ( p = 0.0411); Sed + Veh vs. Exe + Veh ( p = 0.0002); Sed + IL-6 vs. Exe (B6- tub/tub ) ( p = 0.0314); Exe + Veh vs. Exe (ABIL6) ( p = 0.0004); Exe + Veh vs. Exe (B6- tub/tub ) ( p < 0.0001)].

Article Snippet: The following antibodies were also used and previously validated and published ( ; ; ): p-Tyr (PY20) antibody (mouse monoclonal; Santa Cruz Biotechnology cat# sc-508, RRID:AB_628122), JAK2 (HR-758) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-278, RRID: AB_631853), IL-6 (H-183) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-7920, RRID: AB_2127745), and beta-tubulin antibody (rabbit polyclonal; Cell Signaling Technology cat# 2146, RRID: AB_2210545).

Techniques: Western Blot, Expressing, Phospho-proteomics, Standard Deviation, Two Tailed Test

Journal: Frontiers in Physiology

Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus

doi: 10.3389/fphys.2022.956116

Figure Lengend Snippet: Leptin has no additive effect on food intake and TUB signaling in exercised mice or mice treated with IL-6. (A) Evaluation of cumulative food intake (g) at 4 h, 12 h, and 24 h; Western blot showing (B) TUB tyrosine phosphorylation and (C) TUB associated with JAK2 from hypothalamus lysates from C57BL/6J mice (11–12 weeks of age) under resting conditions (Sed) or after swimming exercise (Exe) treated with vehicle or leptin via intracerebroventricular (ICV). In another independent experiment, we also used C57BL/6J mice (11–12 weeks of age) only under resting conditions (Sed) treated with vehicle or IL-6 and leptin via intracerebroventricular (ICV). We analyzed (D) cumulative food intake (g) at 4 h, 12 h, and 24 h; Western blot showing (E) TUB tyrosine phosphorylation and (F) TUB associated with JAK2 from hypothalamus lysates. We used male mice. All values are expressed as means ± standard deviation (SD). Western blot assays ( n = 4 in each group). Two-way ANOVA was used to analyze (A,D) , while one-way ANOVA with Tukey’s multiple comparisons tests was used to analyze (B,C). * Sed Veh vs. Exe Veh, Sed leptin, and Exe leptin ( p < 0.0011); # Sed Veh vs. Exe Veh, Sed leptin, and Exe leptin ( p < 0.0024); & Sed Veh vs. Exe Veh, Sed leptin, and Exe leptin ( p < 0.0012); ϴ Sed Veh vs. IL-6 Veh, Sed leptin, and IL-6 leptin ( p < 0.0017); § Sed Veh vs. IL-6 Veh, Sed leptin, and IL-6 leptin ( p < 0.0001); ϕp<0.0001 vs. other groups.

Article Snippet: The following antibodies were also used and previously validated and published ( ; ; ): p-Tyr (PY20) antibody (mouse monoclonal; Santa Cruz Biotechnology cat# sc-508, RRID:AB_628122), JAK2 (HR-758) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-278, RRID: AB_631853), IL-6 (H-183) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-7920, RRID: AB_2127745), and beta-tubulin antibody (rabbit polyclonal; Cell Signaling Technology cat# 2146, RRID: AB_2210545).

Techniques: Western Blot, Phospho-proteomics, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus

doi: 10.3389/fphys.2022.956116

Figure Lengend Snippet: Effects of IL-6 on food intake and TUB regulation. (A) Evaluation of cumulative food intake (g) at 4 h, 12 h, and 24 h ( n = 5 each group); Western blot showing (B) TUB tyrosine phosphorylation from hypothalamus lysates from young (6–8 weeks of age) heterozygous (B6- tub/+ ) or homozygous (B6- tub/tub ) Tubby mice with vehicle or IL-6 ICV injection. (C) 12 h of cumulative food intake (g) ( n = 5 each group); Western blot showing (D) TUB tyrosine phosphorylation, and (E) TUB associated with JAK2 from hypothalamus lysates from lean C3H/Hepas or C3H/HeJ mice under resting conditions (Sed) or after swimming exercise (Exe). (F) 12 h of cumulative food intake (g) ( n = 5 each group); Western blot showing (G) TUB tyrosine phosphorylation, and (H) TUB associated with JAK2 from hypothalamus lysates from lean C3H/Hepas or C3H/HeJ mice treated with vehicle or IL-6 ICV injection. ICV: intracerebroventricular. We used only male mice. All values are expressed as means ± standard deviation (SD). Western blot assays ( n = 3–5 in each group). Two-way ANOVA with Tukey’s multiple comparisons test was used for analyzing all data. (A) & B6- tub/+ plus Veh vs. B6- tub/+ plus IL-6 ( p = 0.0007); B6- tub/+ plus IL-6 vs. B6- tub/tub plus IL-6 ( p = 0.0012); § B6- tub/+ plus Veh vs. B6- tub/+ plus IL-6 ( p < 0.0068); B6- tub/+ plus IL-6 vs. B6- tub/tub plus Veh or IL-6 ( p < 0.0125). (B) *IL-6 increases TUB phosphorylation independently of genotypes ( p = 0.0006); # B6- tub/+ plus IL-6 vs. B6- tub/tub plus IL-6 ( p = 0.0370). (C) , exercise in the Hepas group decreases food intake compared to other groups ( p < 0.0001). (D,E) exercise in the Hepas group increases TUB phosphorylation and association with JAK2 compared to other groups ( p < 0.0001). (F) IL-6 decreases food intake independently of the genotype compared to sedentary groups ( p < 0.0001). (G,H) IL-6 in the Hepas and Hej groups increases TUB phosphorylation and association with JAK2 compared to the sedentary groups ( p < 0.0001).

Article Snippet: The following antibodies were also used and previously validated and published ( ; ; ): p-Tyr (PY20) antibody (mouse monoclonal; Santa Cruz Biotechnology cat# sc-508, RRID:AB_628122), JAK2 (HR-758) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-278, RRID: AB_631853), IL-6 (H-183) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-7920, RRID: AB_2127745), and beta-tubulin antibody (rabbit polyclonal; Cell Signaling Technology cat# 2146, RRID: AB_2210545).

Techniques: Western Blot, Phospho-proteomics, Injection, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus

doi: 10.3389/fphys.2022.956116

Figure Lengend Snippet: Graphical representation of exercise/IL-6 via the JAK2/TUB axis in the hypothalamus. Acute exercise (swimming) or IL-6 treatment ICV induces TUB tyrosine phosphorylation and its association with JAK2 in the hypothalamus of mice. The activation of the IL-6/Tubby axis reduces food intake. Therefore, acute exercise or IL-6 via the activation of the IL-6/JAK2/Tubby axis reduces feeding.

Article Snippet: The following antibodies were also used and previously validated and published ( ; ; ): p-Tyr (PY20) antibody (mouse monoclonal; Santa Cruz Biotechnology cat# sc-508, RRID:AB_628122), JAK2 (HR-758) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-278, RRID: AB_631853), IL-6 (H-183) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-7920, RRID: AB_2127745), and beta-tubulin antibody (rabbit polyclonal; Cell Signaling Technology cat# 2146, RRID: AB_2210545).

Techniques: Phospho-proteomics, Activation Assay

Journal: Frontiers in Physiology

Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus

doi: 10.3389/fphys.2022.956116

Figure Lengend Snippet: Hypothalamic IL-6 action through JAK2 to activate TUB. (A) Evaluation of cumulative food intake (g) at 4 h, 12 h, and 24 h ( n = 5 in each group); Western blot showing (B) TUB tyrosine phosphorylation and (C) TUB associated with JAK2 from hypothalamic lysates from wt/wt mice (6–8 weeks old) obtained in the colony of B6- tub/tub mice under resting conditions (Sed). The mice were treated with vehicle or AG490 (JAK2inhibitor), IL-6, or IL-6 with AG490 by intracerebroventricular (ICV) injections. We used male mice. All values are expressed as means ± standard deviation (SD). Western blot assays ( n = 4 in each group). Two-way ANOVA was used to analyze A, while one-way ANOVA with Tukey’s multiple comparisons tests was used to analyze B and C. *ICV IL-6 vs. Veh, AG490, and IL-6 plus AG490 ( p < 0.0078); # ICV IL-6 vs. Veh, AG490 and IL-6 plus AG490 ( p < 0.0041); ϕ ICV IL-6 vs. Veh, AG490, and IL-6 plus AG490 ( p < 0.0136).

Article Snippet: The following antibodies were also used and previously validated and published ( ; ; ): p-Tyr (PY20) antibody (mouse monoclonal; Santa Cruz Biotechnology cat# sc-508, RRID:AB_628122), JAK2 (HR-758) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-278, RRID: AB_631853), IL-6 (H-183) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-7920, RRID: AB_2127745), and beta-tubulin antibody (rabbit polyclonal; Cell Signaling Technology cat# 2146, RRID: AB_2210545).

Techniques: Western Blot, Phospho-proteomics, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus

doi: 10.3389/fphys.2022.956116

Figure Lengend Snippet: Effects of TUB knockdown on food intake in response to IL-6 ICV injection. (A) Evaluation of food intake (g) at 4 h, 12 h, and 24 h in lean mice under resting conditions treated ICV with sense; IL-6+ASO; ASO; IL-6+sense ( n = 5 each group). Representative quantification of (B) TUB tyrosine phosphorylation and (C) TUB associated with JAK2 from hypothalamus lysates from lean mice under resting conditions treated with IL-6 ICV or vehicle and pre-treated with ASO against TUB or sense. For this experiment, we used male wt/wt mice (6–8 weeks old) obtained from the B6-tub/tub colony. All values are expressed as means ± standard deviation (SD). Western blot assays ( n = 3–4 in each group). Two-way ANOVA was used to analyze A, and one-way ANOVA with Tukey’s multiple comparisons tests was used to analyze B and C. * indicates IL-6 vs. Sense, ASO, and ASO plus IL-6 ( p < 0.0303); # indicates IL-6 vs. Sense, ASO, and ASO plus IL-6 ( p < 0.0042); § indicates Sense vs. ASO, and ASO plus IL-6 ( p <0.0198); & indicates IL-6 vs. Sense, ASO,and ASO plus IL-6 ( p < 0.0092); ϴ indicates Sense vs. ASO, and ASO plus IL-6 ( p < 0.0400).

Article Snippet: The following antibodies were also used and previously validated and published ( ; ; ): p-Tyr (PY20) antibody (mouse monoclonal; Santa Cruz Biotechnology cat# sc-508, RRID:AB_628122), JAK2 (HR-758) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-278, RRID: AB_631853), IL-6 (H-183) antibody (rabbit polyclonal; Santa Cruz Biotechnology cat# sc-7920, RRID: AB_2127745), and beta-tubulin antibody (rabbit polyclonal; Cell Signaling Technology cat# 2146, RRID: AB_2210545).

Techniques: Knockdown, Injection, Phospho-proteomics, Standard Deviation, Western Blot

Journal: Cell Death and Differentiation

Article Title: The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma

doi: 10.1038/s41418-019-0386-6

Figure Lengend Snippet: p140Cap affects signaling pathways in NB cells. Cell extracts were run on 4–15% pre-casted SDS-PAGE and analyzed as follows. a Mock and p140 ACN cells. Src activation was evaluated by WB analysis of Tyr 416 phosphorylation (Y416) and Src protein level as loading control. Antibodies to GAPDH (as loading control) were used. Quantification on the right is the ratio between phosphorylated Src and total Src protein from three independent experiment, as mean ± SEM (right) (unpaired t -test ** P < 0.01). b Mock and p140 ACN cells. p130Cas phosphorylation was evaluated with antibodies to phosphorylated p130Cas at Tyr 410, p130Cas and GAPDH antibodies for loading control. Quantification on the right is the ratio between phosphorylated p130Cas and total p130Cas protein from three independent experiment, as mean ± SEM (right) (unpaired t -test *** P < 0.001). c Mock and p140 ACN cells. STAT3 phosphorylation was evaluated with antibodies to phosphorylated STAT3 at Tyr705 and STAT3 antibodies for loading control. Quantification on the right is the ratio between phosphorylated STAT3 and total STAT3 protein from three independent experiment, as mean ± SEM (right) (unpaired t -test *** P < 0.001). d Mock and p140 ACN cells. Jak2 activation was evaluated with antibodies to phosphorylated Jak2 at Tyr1007/1008) and Jak2 antibodies for loading control. Quantification on the right is the ratio between phosphorylated Jak2 and total Jak2 protein from three independent experiment, as mean ± SEM (right) (unpaired t -test *** P < 0.001). e–g SH-5Y-SY cells were transfected with a pool of p140Cap specific siRNA (si-p140) or the appropriate siRNA control (Ctr). At 72 h cells were extracted and tested for p140Cap WB, and GAPDH as loading contro ( e ), for Src activation at Tyr 416 phosphorylation (Y416) and Src protein level as loading control ( f ), and for phosphorylated STAT3 at Tyr 705 and STAT3 protein level as loading control ( g ). Quantification is shown on the left as the ratio between phosphorylated Src/STAT3 and total Src/STAT3 protein from three independent experiment, as mean ± SEM (right) (unpaired t -test * P < 0.05; ** P < 0.01). h Bcl2 expression upon apoptotic stimulus was evaluated by WB of Mock and p140 cells, kept in suspension for 12 h (apoptotic stimulus) or in standard culture (basal conditions). Vinculin antibodies were used for loading controls. Quantification on the right is the ratio between Bcl2 and Vinculin protein from three independent experiment, as mean ± SEM (unpaired t -test *** P < 0.001). i Annexin V levels were assessed by a Flow cytometry assay to detect the percentage (%) of apoptotic cells in mock and p140 cells, kept in suspension for 24 h (apoptotic stimulus) or in standard culture (basal conditions) (unpaired t -test * P < 0.05). j p140 cells in apoptotic stimulus as in H were evaluated for STAT3 phosphorylation. Quantification on the right is the ratio between phosphorylated STAT3 (pSTAT3) and total STAT3 protein from three independent experiment, as mean ± SEM (unpaired t -test * P < 0.05)

Article Snippet: The following antibodies were used: mouse monoclonal antibodies (Mab) to p140Cap already characterized in [ ] (1:500), anti phospho-p130Cas (Tyr410; #4011, 1:1000), anti phospho-Src (Tyr416; #2101, 1:1000), anti phospho-JAK2 (Tyr1007/1008; #3776S, 1:1000), anti γH2AX (Ser139; #2577, 1:1000) and anti H2AX (#2595, 1:1000) from Cell Signaling, Beverly, MA; anti GAPDH (MAB374, 1:8000) from Millipore, Billerica, MA, USA; anti p130Cas (cat.no 610272, 1:2500) from BD Transduction Laboratories, Franklin Lakes, NY; anti Src (B-12, 1:1000) and anti JAK2 (sc-278, 1:1000) from Santa Cruz Biotechnologies, Palo Alto, CA, USA; anti Tubulin (T5168, 1:8000) from Sigma-Aldrich Co, Italy.

Techniques: Protein-Protein interactions, SDS Page, Activation Assay, Phospho-proteomics, Control, Transfection, Expressing, Suspension, Flow Cytometry

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