jak2 hr 758 antibody Search Results


jak2  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology jak2
Jak2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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jak2 hr 758 antibody  (Santa Cruz Biotechnology)
96
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 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti nucleolin antibody  (Danaher Inc)
99
Danaher Inc rabbit polyclonal anti nucleolin antibody
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Rabbit Polyclonal Anti Nucleolin Antibody, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mcm5 antibody  (NSJ Bioreagents)
99
NSJ Bioreagents mcm5 antibody
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Mcm5 Antibody, supplied by NSJ Bioreagents, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti irs-1/2  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology anti irs-1/2
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Anti Irs 1/2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti mouse jak 1  (Upstate Biotechnology Inc)
86
Upstate Biotechnology Inc anti mouse jak 1
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Anti Mouse Jak 1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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polyclonal anti stat3  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology polyclonal anti stat3
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Polyclonal Anti Stat3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti akt1  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology anti akt1
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Anti Akt1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti stat1  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology anti stat1
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Anti Stat1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti tyr p  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology anti tyr p
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Anti Tyr P, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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antiphosphotyrosine  (Transduction Laboratories Inc)
90
Transduction Laboratories Inc antiphosphotyrosine
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Antiphosphotyrosine, supplied by Transduction Laboratories Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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monoclonal anti gst antibody  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology monoclonal anti gst antibody
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit <t>polyclonal</t> antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal <t>anti-nucleolin</t> antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm
Monoclonal Anti Gst Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


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

Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit polyclonal antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal anti-nucleolin antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm

Journal: Retrovirology

Article Title: The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag

doi: 10.1186/s12977-018-0428-z

Figure Lengend Snippet: Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag 1-200- RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag 1-200 -GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit polyclonal antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (* p < 0.05; ** p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O 2 , 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal anti-nucleolin antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. b. Scale bar represents 10 µm

Article Snippet: At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal anti-nucleolin antibody (ab 22,758, Abcam, 1:800).

Techniques: Infection, Stable Transfection, Expressing, Staining, Flow Cytometry, Produced, Standard Deviation, Comparison

GRI and GRIII boxes of PFV Gag are Nucleolar Localization Signals. a Scheme of PFV Gag protein where the primary protease-cleavage site at residue 621 is indicated by a dotted line. Some characterized motifs are shown. CTRS: cytoplasmic targeting and retention signal (aa 43–60); NES: nuclear export signal (aa 95–112); dim: dimerization domain (aa 130–160); GRI, GRII and GRIII: glycine-arginine rich box I (aa 485–511), II (aa 534–557) and III (aa 586–618); CBM, chromatin-binding motif (aa 536–544). b The subcellular localization of GRI, GRII, GRII R540A or GRIII expressed as GFP-fusion proteins in fixed HeLa cells was analyzed 24 h post-transfection by immunofluorescence and confocal microscopy. Nucleoli were immune-stained with an anti-nucleolin antibody (ab 22758, Abcam, 1:800) and nuclei were stained with DAPI (blue). c The localization of GFP-GRI expressed in HeLa cells relative to the NoLS of HIV-1 Rev protein (aa 35–51) in fusion with DsRed or specific markers of the nucleolar subcompartments was studied as in B. Cells were stained with antibodies against fibrillarin (c13c3, Cell signaling, 1:200), B23 (sc6013_R, Santa Cruz, 1:200) or UBF (H300, Santa Cruz, 1:200) to visualize the dense fibrillar component (DFC), the granular component (GC) and the fibrillar center (FC), respectively. The right column (zoom × 16) corresponds to the enlarged images from the boxed areas. Scale bar represents 10 µm

Journal: Retrovirology

Article Title: The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag

doi: 10.1186/s12977-018-0428-z

Figure Lengend Snippet: GRI and GRIII boxes of PFV Gag are Nucleolar Localization Signals. a Scheme of PFV Gag protein where the primary protease-cleavage site at residue 621 is indicated by a dotted line. Some characterized motifs are shown. CTRS: cytoplasmic targeting and retention signal (aa 43–60); NES: nuclear export signal (aa 95–112); dim: dimerization domain (aa 130–160); GRI, GRII and GRIII: glycine-arginine rich box I (aa 485–511), II (aa 534–557) and III (aa 586–618); CBM, chromatin-binding motif (aa 536–544). b The subcellular localization of GRI, GRII, GRII R540A or GRIII expressed as GFP-fusion proteins in fixed HeLa cells was analyzed 24 h post-transfection by immunofluorescence and confocal microscopy. Nucleoli were immune-stained with an anti-nucleolin antibody (ab 22758, Abcam, 1:800) and nuclei were stained with DAPI (blue). c The localization of GFP-GRI expressed in HeLa cells relative to the NoLS of HIV-1 Rev protein (aa 35–51) in fusion with DsRed or specific markers of the nucleolar subcompartments was studied as in B. Cells were stained with antibodies against fibrillarin (c13c3, Cell signaling, 1:200), B23 (sc6013_R, Santa Cruz, 1:200) or UBF (H300, Santa Cruz, 1:200) to visualize the dense fibrillar component (DFC), the granular component (GC) and the fibrillar center (FC), respectively. The right column (zoom × 16) corresponds to the enlarged images from the boxed areas. Scale bar represents 10 µm

Article Snippet: At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal anti-nucleolin antibody (ab 22,758, Abcam, 1:800).

Techniques: Residue, Binding Assay, Transfection, Immunofluorescence, Confocal Microscopy, Staining

PRMT-1 binds to and methylates PFV Gag in a manner that depends on R540. a Following lysis, cells expressing PFV Gag WT or R450A mutant and GFP-PRMT1 were incubated with protein A beads coated with either an anti-GFP (cat.11 814 460 001, Roche, 1:100) or an anti-ADMA (ab5394 (7E6), Abcam,1:100) antibody. Input and immunoprecipitated proteins were separated by SDS-PAGE and visualized by Western blotting with anti-GFP (cat.11 814 460 001, Roche, 1:1000) or rabbit polyclonal anti-PFV antibodies. b Lysates from cells expressing HA-tagged GRs or GRs-R540A were immunoprecipitated with an antibody directed against PRMT1 (Cat A300-722A, Bethyl Laboratories (Euromedex), 1:100), the ADMA modification (ab5394 (7E6), Abcam, 1:100), or the HA epitope (H11, Covance, 1:100). Input and bound proteins were analyzed as in A. c HeLa cells were transfected with siRNA targeting PRMT1 or scrambled control (scr) and, two days later, with GFP-GRs expression plasmid. After 24 h, cells were processed as indicated in Fig. b. Images are representative of two independent experiments. The numbers indicate the percentage of GFP-positive cells with significant nucleolar accumulation of 100 counted cells. Scale bar represents 10 µm

Journal: Retrovirology

Article Title: The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag

doi: 10.1186/s12977-018-0428-z

Figure Lengend Snippet: PRMT-1 binds to and methylates PFV Gag in a manner that depends on R540. a Following lysis, cells expressing PFV Gag WT or R450A mutant and GFP-PRMT1 were incubated with protein A beads coated with either an anti-GFP (cat.11 814 460 001, Roche, 1:100) or an anti-ADMA (ab5394 (7E6), Abcam,1:100) antibody. Input and immunoprecipitated proteins were separated by SDS-PAGE and visualized by Western blotting with anti-GFP (cat.11 814 460 001, Roche, 1:1000) or rabbit polyclonal anti-PFV antibodies. b Lysates from cells expressing HA-tagged GRs or GRs-R540A were immunoprecipitated with an antibody directed against PRMT1 (Cat A300-722A, Bethyl Laboratories (Euromedex), 1:100), the ADMA modification (ab5394 (7E6), Abcam, 1:100), or the HA epitope (H11, Covance, 1:100). Input and bound proteins were analyzed as in A. c HeLa cells were transfected with siRNA targeting PRMT1 or scrambled control (scr) and, two days later, with GFP-GRs expression plasmid. After 24 h, cells were processed as indicated in Fig. b. Images are representative of two independent experiments. The numbers indicate the percentage of GFP-positive cells with significant nucleolar accumulation of 100 counted cells. Scale bar represents 10 µm

Article Snippet: At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal anti-nucleolin antibody (ab 22,758, Abcam, 1:800).

Techniques: Lysis, Expressing, Mutagenesis, Incubation, Immunoprecipitation, SDS Page, Western Blot, Modification, Transfection, Control, Plasmid Preparation