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jak2 hr 758 antibody  (Santa Cruz Biotechnology)


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    Santa Cruz Biotechnology jak2 hr 758 antibody
    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 <t>JAK2</t> 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)].
    Jak2 Hr 758 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 2434 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/jak2+hr+758+antibody/p-Tyr+Antibody/pmc09702993-86-26-31
    Average 96 stars, based on 2434 article reviews
    jak2 hr 758 antibody - by Bioz Stars, 2026-09
    96/100 stars

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    1) Product Images from "Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus"

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

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2022.956116

    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)].
    Figure Legend 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)].

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Phospho-proteomics, Activation Assay

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    Related Articles

    Western Blot:

    Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus
    Article Snippet: similar antibody from Santa Cruz against C-terminal was previously validated and published ( ; ; ). .. 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). .. We used UN-SCAN-IT gel software to determine the band intensities.

    Expressing:

    Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus
    Article Snippet: similar antibody from Santa Cruz against C-terminal was previously validated and published ( ; ; ). .. 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). .. We used UN-SCAN-IT gel software to determine the band intensities.

    Phospho-proteomics:

    Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus
    Article Snippet: similar antibody from Santa Cruz against C-terminal was previously validated and published ( ; ; ). .. 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). .. We used UN-SCAN-IT gel software to determine the band intensities.

    Standard Deviation:

    Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus
    Article Snippet: similar antibody from Santa Cruz against C-terminal was previously validated and published ( ; ; ). .. 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). .. We used UN-SCAN-IT gel software to determine the band intensities.

    Two Tailed Test:

    Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus
    Article Snippet: similar antibody from Santa Cruz against C-terminal was previously validated and published ( ; ; ). .. 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). .. We used UN-SCAN-IT gel software to determine the band intensities.

    Injection:

    Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus
    Article Snippet: similar antibody from Santa Cruz against C-terminal was previously validated and published ( ; ; ). .. 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). .. We used UN-SCAN-IT gel software to determine the band intensities.

    Activation Assay:

    Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus
    Article Snippet: similar antibody from Santa Cruz against C-terminal was previously validated and published ( ; ; ). .. 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). .. We used UN-SCAN-IT gel software to determine the band intensities.

    Knockdown:

    Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus
    Article Snippet: similar antibody from Santa Cruz against C-terminal was previously validated and published ( ; ; ). .. 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). .. We used UN-SCAN-IT gel software to determine the band intensities.



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    Santa Cruz Biotechnology polyclonal anti jak2 hr 758 antibody
    FIG. 1. OB-R fusion protein constructs. Schematic representation of the fusion proteins used in this study. The short (OB-Rs) and the long (OB-Rl) OB-R isoforms were fused at their C-terminal tails with the BRET partners (Luc and YFP) to monitor basal and ligand-induced BRET. BOX1, <t>JAK2-binding</t> site including the PNP motif (amino acid residues, one-letter code).
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    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)].

    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

    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.

    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

    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).

    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

    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.

    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

    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).

    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

    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).

    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

    KEY RESOURCES TABLE

    Journal: Cell

    Article Title: Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis

    doi: 10.1016/j.cell.2018.02.036

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit polyclonal anti-JAK2 (HR-758) , Santa Cruz Biotechnology , Cat#: sc-278; RRID: AB_631853.

    Techniques: Recombinant, Modification, Clinical Proteomics, Transfection, Infection, Protease Inhibitor, Mutagenesis, cDNA Synthesis, SYBR Green Assay, Western Blot, Lysis, Isolation, PCR Cloning, Sequencing, Mass Spectrometry, Flow Cytometry, Clone Assay, Software

    FIG. 1. OB-R fusion protein constructs. Schematic representation of the fusion proteins used in this study. The short (OB-Rs) and the long (OB-Rl) OB-R isoforms were fused at their C-terminal tails with the BRET partners (Luc and YFP) to monitor basal and ligand-induced BRET. BOX1, JAK2-binding site including the PNP motif (amino acid residues, one-letter code).

    Journal: The Journal of biological chemistry

    Article Title: Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers.

    doi: 10.1074/jbc.M302002200

    Figure Lengend Snippet: FIG. 1. OB-R fusion protein constructs. Schematic representation of the fusion proteins used in this study. The short (OB-Rs) and the long (OB-Rl) OB-R isoforms were fused at their C-terminal tails with the BRET partners (Luc and YFP) to monitor basal and ligand-induced BRET. BOX1, JAK2-binding site including the PNP motif (amino acid residues, one-letter code).

    Article Snippet: The cells were scraped in lysis buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 5% glycerol, 0.02% NaN3, 0.1% Nonidet P-40, 1 mM orthovanadate, 5 mg/liter soybean trypsin inhibitor, and 10 mg/liter benzamidine) and centrifuged for 15 min at 18,000 g. The soluble fraction was subjected to immunoprecipitation for 2 h with a polyclonal anti-JAK2 (HR-758) antibody (1 g/ml) (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Construct, Binding Assay

    FIG. 2. Characterization of OB-R fusion proteins. A, surface expression of OB-R constructs in COS-7 cells estimated by radioligand binding studies using 125I-leptin as radioligand. Surface expressed re- ceptors are expressed as percentages of total receptors determined as described under “Experimental Procedures.” The data are the means S.E. of at least three independent experiments performed in duplicate. B, the localization of the indicated YFP proteins was assessed by fluo- rescence microscopy in COS-7 cells. C, activation of HA-JAK2 by the indicated OB-R constructs in HeLa cells in the presence of leptin. The total lysates were immunoprecipitated with anti-JAK2 antibodies, and JAK2 autophosphorylation on tyrosine residues was detected by immu- noblotting using 4G10 anti-phosphotyrosine antibody. Similar results were obtained in two further experiments. D, activation of a STAT3 reporter gene in HeLa cells by the indicated OB-R constructs in the presence of 10 nM leptin. The data are the means S.E. of at least three independent experiments and are presented as fold stimulation by leptin. NT, non-transfected; IP, immunoprecipitation; IB, immunoblot; wt, wild type.

    Journal: The Journal of biological chemistry

    Article Title: Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers.

    doi: 10.1074/jbc.M302002200

    Figure Lengend Snippet: FIG. 2. Characterization of OB-R fusion proteins. A, surface expression of OB-R constructs in COS-7 cells estimated by radioligand binding studies using 125I-leptin as radioligand. Surface expressed re- ceptors are expressed as percentages of total receptors determined as described under “Experimental Procedures.” The data are the means S.E. of at least three independent experiments performed in duplicate. B, the localization of the indicated YFP proteins was assessed by fluo- rescence microscopy in COS-7 cells. C, activation of HA-JAK2 by the indicated OB-R constructs in HeLa cells in the presence of leptin. The total lysates were immunoprecipitated with anti-JAK2 antibodies, and JAK2 autophosphorylation on tyrosine residues was detected by immu- noblotting using 4G10 anti-phosphotyrosine antibody. Similar results were obtained in two further experiments. D, activation of a STAT3 reporter gene in HeLa cells by the indicated OB-R constructs in the presence of 10 nM leptin. The data are the means S.E. of at least three independent experiments and are presented as fold stimulation by leptin. NT, non-transfected; IP, immunoprecipitation; IB, immunoblot; wt, wild type.

    Article Snippet: The cells were scraped in lysis buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 5% glycerol, 0.02% NaN3, 0.1% Nonidet P-40, 1 mM orthovanadate, 5 mg/liter soybean trypsin inhibitor, and 10 mg/liter benzamidine) and centrifuged for 15 min at 18,000 g. The soluble fraction was subjected to immunoprecipitation for 2 h with a polyclonal anti-JAK2 (HR-758) antibody (1 g/ml) (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Construct, Binding Assay, Microscopy, Activation Assay, Immunoprecipitation, Transfection, Western Blot

    FIG. 5. Role of JAK2 in OB-Rs dimerization. A, activation of JAK2 by OB-Rs wild type (wt) and Box 1 SNS mutant constructs. HeLa cells co-expressing HA-JAK2 and the indicated OB-R constructs were prein- cubated for 1 h in the absence or presence of 5 nM AG490 and then stimulated with 100 nM leptin for 5 min. The total lysates were immu- noprecipitated (IP) with anti-JAK2 antibodies, and JAK2 autophospho- rylation on tyrosine residues was detected by immunoblotting (IB) using 4G10 anti-phosphotyrosine antibody. B, HeLa cells co-expressing Luc and YFP fusion proteins of the indicated OB-R at a 1:1 protein ratio were preincubated as in A. The cells were detached, and BRET meas- urements were performed as described in the legend to Fig. 3A. C, HeLa cells co-expressing OB-Rs-Luc and OB-Rs-YFP wild type (E) or SNS mutants (G) at a 1:1 protein ratio of the two BRET partners were preincubated for 5 min with increasing concentrations of human leptin in the presence of 0.015% saponin before BRET measurements. The data are the means S.E. of at least three independent experiments, each performed in duplicate.

    Journal: The Journal of biological chemistry

    Article Title: Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers.

    doi: 10.1074/jbc.M302002200

    Figure Lengend Snippet: FIG. 5. Role of JAK2 in OB-Rs dimerization. A, activation of JAK2 by OB-Rs wild type (wt) and Box 1 SNS mutant constructs. HeLa cells co-expressing HA-JAK2 and the indicated OB-R constructs were prein- cubated for 1 h in the absence or presence of 5 nM AG490 and then stimulated with 100 nM leptin for 5 min. The total lysates were immu- noprecipitated (IP) with anti-JAK2 antibodies, and JAK2 autophospho- rylation on tyrosine residues was detected by immunoblotting (IB) using 4G10 anti-phosphotyrosine antibody. B, HeLa cells co-expressing Luc and YFP fusion proteins of the indicated OB-R at a 1:1 protein ratio were preincubated as in A. The cells were detached, and BRET meas- urements were performed as described in the legend to Fig. 3A. C, HeLa cells co-expressing OB-Rs-Luc and OB-Rs-YFP wild type (E) or SNS mutants (G) at a 1:1 protein ratio of the two BRET partners were preincubated for 5 min with increasing concentrations of human leptin in the presence of 0.015% saponin before BRET measurements. The data are the means S.E. of at least three independent experiments, each performed in duplicate.

    Article Snippet: The cells were scraped in lysis buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 5% glycerol, 0.02% NaN3, 0.1% Nonidet P-40, 1 mM orthovanadate, 5 mg/liter soybean trypsin inhibitor, and 10 mg/liter benzamidine) and centrifuged for 15 min at 18,000 g. The soluble fraction was subjected to immunoprecipitation for 2 h with a polyclonal anti-JAK2 (HR-758) antibody (1 g/ml) (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Activation Assay, Mutagenesis, Construct, Expressing, Western Blot