anti ghrelin receptor ghsr extracellular antibody  (Becton Dickinson)

 
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    Becton Dickinson anti ghrelin receptor ghsr extracellular antibody
    Roles of rpS6 kinases and PP2A in <t>ghrelin-induced</t> activation of eEF2 and protein synthesis. a The schematic of HEK293 GHS-R1α-EGFP+ cells treatment. b Changes in eEF2 and eEF2K phosphorylation induced by ghrelin after inhibition of either p90 RSK (10 µM BRD7389) or p70 S6K (1 µM PF4708671). c Effects of ghrelin on p-eEF2 (Thr56) and p-eEF2K (Ser366 and Ser500) levels in cells upon inhibition of p90 RSK and p70 S6K (double-treatment with BRD7389/PF4708671) or PP2A (OKA, 50 nM). In ( b , c ), cells were pre-treated with kinase or phosphatase inhibitors for 2 h prior to ghrelin addition (100 nM, 30 min). Changes in p-mTOR and p-ERK levels were assessed to confirm the GHS-R1α activation. d Changes in SNAP and G Luc production in cells treated with ghrelin upon inhibition of p70 S6K (1 µM PF4708671). Cells were pre-treated for 2 h and then treated with ghrelin (100 nM, 30 min) in the presence of PF4708671; an arrow shows the residual amounts of SNAP in cells lysed immediately after their treatment with SNAP block. e Comparative analysis of eEF2 response to ghrelin in resting and OKA-treated cells [extracted from ( c )]. The bottom panel shows that the pre-treatment with OKA causes an increase in phosphorylation of ERK, a common target of PP2A. Red rectangles ( b , c ) highlight effects of BRD7389 on total levels of mTOR, eEF2 and eEF2K proteins. Red asterisks indicate significant difference in the p-eEF2 (Thr56) response to ghrelin in mock-treated vs BRD- and PF-treated cells ( b ), or in mock-treated vs BRD/PF- and OKA-treated cells ( c ), red hash signs—significant difference in p-eEF2 response to ghrelin between BRD/PF- and OKA-treated cells. Black asterisks show the same for p-eEF2K (Ser366) levels. Columns/horizontal bars and gradient circles show mean values and individual data points, respectively. Statistical details: b effects of p90 RSK or p70 S6K inhibition on the ghrelin-induced changes in eEF2 and eEF2K phosphorylation; (a) p-eEF2 (2,6) = 29.442, p
    Anti Ghrelin Receptor Ghsr Extracellular Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ghrelin receptor ghsr extracellular antibody/product/Becton Dickinson
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ghrelin receptor ghsr extracellular antibody - by Bioz Stars, 2022-10
    86/100 stars

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    1) Product Images from "Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis"

    Article Title: Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-022-04446-4

    Roles of rpS6 kinases and PP2A in ghrelin-induced activation of eEF2 and protein synthesis. a The schematic of HEK293 GHS-R1α-EGFP+ cells treatment. b Changes in eEF2 and eEF2K phosphorylation induced by ghrelin after inhibition of either p90 RSK (10 µM BRD7389) or p70 S6K (1 µM PF4708671). c Effects of ghrelin on p-eEF2 (Thr56) and p-eEF2K (Ser366 and Ser500) levels in cells upon inhibition of p90 RSK and p70 S6K (double-treatment with BRD7389/PF4708671) or PP2A (OKA, 50 nM). In ( b , c ), cells were pre-treated with kinase or phosphatase inhibitors for 2 h prior to ghrelin addition (100 nM, 30 min). Changes in p-mTOR and p-ERK levels were assessed to confirm the GHS-R1α activation. d Changes in SNAP and G Luc production in cells treated with ghrelin upon inhibition of p70 S6K (1 µM PF4708671). Cells were pre-treated for 2 h and then treated with ghrelin (100 nM, 30 min) in the presence of PF4708671; an arrow shows the residual amounts of SNAP in cells lysed immediately after their treatment with SNAP block. e Comparative analysis of eEF2 response to ghrelin in resting and OKA-treated cells [extracted from ( c )]. The bottom panel shows that the pre-treatment with OKA causes an increase in phosphorylation of ERK, a common target of PP2A. Red rectangles ( b , c ) highlight effects of BRD7389 on total levels of mTOR, eEF2 and eEF2K proteins. Red asterisks indicate significant difference in the p-eEF2 (Thr56) response to ghrelin in mock-treated vs BRD- and PF-treated cells ( b ), or in mock-treated vs BRD/PF- and OKA-treated cells ( c ), red hash signs—significant difference in p-eEF2 response to ghrelin between BRD/PF- and OKA-treated cells. Black asterisks show the same for p-eEF2K (Ser366) levels. Columns/horizontal bars and gradient circles show mean values and individual data points, respectively. Statistical details: b effects of p90 RSK or p70 S6K inhibition on the ghrelin-induced changes in eEF2 and eEF2K phosphorylation; (a) p-eEF2 (2,6) = 29.442, p
    Figure Legend Snippet: Roles of rpS6 kinases and PP2A in ghrelin-induced activation of eEF2 and protein synthesis. a The schematic of HEK293 GHS-R1α-EGFP+ cells treatment. b Changes in eEF2 and eEF2K phosphorylation induced by ghrelin after inhibition of either p90 RSK (10 µM BRD7389) or p70 S6K (1 µM PF4708671). c Effects of ghrelin on p-eEF2 (Thr56) and p-eEF2K (Ser366 and Ser500) levels in cells upon inhibition of p90 RSK and p70 S6K (double-treatment with BRD7389/PF4708671) or PP2A (OKA, 50 nM). In ( b , c ), cells were pre-treated with kinase or phosphatase inhibitors for 2 h prior to ghrelin addition (100 nM, 30 min). Changes in p-mTOR and p-ERK levels were assessed to confirm the GHS-R1α activation. d Changes in SNAP and G Luc production in cells treated with ghrelin upon inhibition of p70 S6K (1 µM PF4708671). Cells were pre-treated for 2 h and then treated with ghrelin (100 nM, 30 min) in the presence of PF4708671; an arrow shows the residual amounts of SNAP in cells lysed immediately after their treatment with SNAP block. e Comparative analysis of eEF2 response to ghrelin in resting and OKA-treated cells [extracted from ( c )]. The bottom panel shows that the pre-treatment with OKA causes an increase in phosphorylation of ERK, a common target of PP2A. Red rectangles ( b , c ) highlight effects of BRD7389 on total levels of mTOR, eEF2 and eEF2K proteins. Red asterisks indicate significant difference in the p-eEF2 (Thr56) response to ghrelin in mock-treated vs BRD- and PF-treated cells ( b ), or in mock-treated vs BRD/PF- and OKA-treated cells ( c ), red hash signs—significant difference in p-eEF2 response to ghrelin between BRD/PF- and OKA-treated cells. Black asterisks show the same for p-eEF2K (Ser366) levels. Columns/horizontal bars and gradient circles show mean values and individual data points, respectively. Statistical details: b effects of p90 RSK or p70 S6K inhibition on the ghrelin-induced changes in eEF2 and eEF2K phosphorylation; (a) p-eEF2 (2,6) = 29.442, p

    Techniques Used: Activation Assay, Inhibition, Blocking Assay

    Susceptibility of ghrelin-induced de-suppression of eEF2 to metabolic stress. a The schematic of experiments and total ATP analysis in HEK293 GHS-R1α-EGFP+ cells supplied with or deprived of glucose for 15 h. b Western blotting analysis of changes in eEF2, eEF2K, mTOR and ERK phosphorylation induced by ghrelin treatment (50 nM and 100 nM, 30 min). c Glucose concentration-dependent changes of p-eEF2 and p-eEF2K levels in response to ghrelin (100 nM, 30 min), Western blotting analysis. The quantification curve shows dynamics of p-eEF2 levels, and include values corresponding to 1 mM glucose concentration (see Fig. S5a, c); maximal decrease in eEF2 phosphorylation corresponding to the ‘physiological’ range of glucose concentrations (2–10 mM) is highlighted by a rectangle. d Levels of eIF4G1 and 4E-BP1 proteins associated with the cap binding complex in resting and metabolically stressed cells stimulated with mock (DMEM) and 100 nM ghrelin for 30 min; in glucose-deprived cells the levels of the cap-bound initiation inhibitor 4E-BP1 strongly increase. e Effect of ghrelin (50 nM, 15 and 30 min) on protein phosphorylation in cells pre-treated for 2 h with FCCP (mitochondrial uncoupler, 1 µM) and quantitative analysis of p-eEF2 levels. Data are presented as mean ± SD. Gradient circles demonstrate individual data points. Significant difference from mock ( c , e ) and between glucose (+) and galactose (−) samples ( b ) is shown by asterisks. Statistical details: b effect of glucose deprivation on the ghrelin-induced changes in protein phosphorylation (50 nM ghrelin), N = 3, independent samples T test: p-mTOR t (4) = 2.109, p = 0.103; p-eEF2 t (4) = − 3.939, p = 0.017; p-eEF2K t (4) = 5.001, p = 0.007. c Effect of glucose concentration on the response to ghrelin: (a) p-eEF2 (4,8) = 28.243, p
    Figure Legend Snippet: Susceptibility of ghrelin-induced de-suppression of eEF2 to metabolic stress. a The schematic of experiments and total ATP analysis in HEK293 GHS-R1α-EGFP+ cells supplied with or deprived of glucose for 15 h. b Western blotting analysis of changes in eEF2, eEF2K, mTOR and ERK phosphorylation induced by ghrelin treatment (50 nM and 100 nM, 30 min). c Glucose concentration-dependent changes of p-eEF2 and p-eEF2K levels in response to ghrelin (100 nM, 30 min), Western blotting analysis. The quantification curve shows dynamics of p-eEF2 levels, and include values corresponding to 1 mM glucose concentration (see Fig. S5a, c); maximal decrease in eEF2 phosphorylation corresponding to the ‘physiological’ range of glucose concentrations (2–10 mM) is highlighted by a rectangle. d Levels of eIF4G1 and 4E-BP1 proteins associated with the cap binding complex in resting and metabolically stressed cells stimulated with mock (DMEM) and 100 nM ghrelin for 30 min; in glucose-deprived cells the levels of the cap-bound initiation inhibitor 4E-BP1 strongly increase. e Effect of ghrelin (50 nM, 15 and 30 min) on protein phosphorylation in cells pre-treated for 2 h with FCCP (mitochondrial uncoupler, 1 µM) and quantitative analysis of p-eEF2 levels. Data are presented as mean ± SD. Gradient circles demonstrate individual data points. Significant difference from mock ( c , e ) and between glucose (+) and galactose (−) samples ( b ) is shown by asterisks. Statistical details: b effect of glucose deprivation on the ghrelin-induced changes in protein phosphorylation (50 nM ghrelin), N = 3, independent samples T test: p-mTOR t (4) = 2.109, p = 0.103; p-eEF2 t (4) = − 3.939, p = 0.017; p-eEF2K t (4) = 5.001, p = 0.007. c Effect of glucose concentration on the response to ghrelin: (a) p-eEF2 (4,8) = 28.243, p

    Techniques Used: Western Blot, Concentration Assay, Binding Assay, Metabolic Labelling

    Changes in protein synthesis in HEK293 GHS-R1α-EGFP+ cells in response to GHS-R1α activation by ghrelin. a Efficiency of cell transfection with a plasmid encoding SNAP ( > 55% SNAP-positive cells 24 h after the onset of transfection). b Representative fluorescent image of the gel showing the amounts of SNAP protein (647-SiR fluorescence) produced in cells treated with 100 nM ghrelin for 0–60 min. c The ratio of SNAP production in cells treated with ghrelin or mock (medium) for 0–240 min, corresponding to ( b ). d Dynamics of G Luc production by cells treated with 100 nM ghrelin. The ratio of G Luc signals in the supernatants of cells treated with ghrelin or mock (medium) for indicated time. Note that over 90% of the newly produced G Luc is rapidly secreted from cells, thus minimising risks of intracellular protein degradation. e Signal-to-noise ratios calculated for the 15-min time point vs 0 time point (no ghrelin) of G Luc- and SNAP-based analysis. f RT-qPCR analysis of SNAP and G Luc mRNA levels in cells transfected with the corresponding plasmids and treated with mock or 100 nM ghrelin for 30 min ( N = 2). g Representative images (stacks of 7 focal planes) of Alexa Fluor 594 and EGFP fluorescence (showing nascent peptides (in red) and GHS-R1α (in blue), respectively) in mock and ghrelin-treated cells (100 nM ghrelin for 30 min); Click-iT ® HPG Alexa Fluor 594 Protein Synthesis Assay Kit was used. h Quantitative analysis of the Alexa Fluor 594 intensity signals in ghrelin- and mock-treated cells. i , j Distribution of ribosomes in sucrose density gradients and monosome/polysome (M/P) ratio in HEK293 GHS-R1α-EGFP+ cells treated with 100 nM ghrelin for 0–60 min ( N = 3). Error bars in ( c , d ) are presented for experiments repeated at least three times. In ( e , h ), boxes show median values and the interquartile range; in ( e ), whiskers extend to the minimal and maximal values; in ( h ), Altman-style whiskers extend to 5th and 95th percentiles, and all analysed cells are represented by data points ( n > 90 in each group, N = 3 independent experiments). Data are shown as individual data points (circles in f and j ; dots in h ), mean values (horizontal bars, f, h and j ) and mean ± SD. Asterisks show significant difference between ghrelin- and mock-treated (0-time point) cells. Statistical details: b, c Effect of ghrelin treatment time: SNAP (5, 15) = 27.048, p
    Figure Legend Snippet: Changes in protein synthesis in HEK293 GHS-R1α-EGFP+ cells in response to GHS-R1α activation by ghrelin. a Efficiency of cell transfection with a plasmid encoding SNAP ( > 55% SNAP-positive cells 24 h after the onset of transfection). b Representative fluorescent image of the gel showing the amounts of SNAP protein (647-SiR fluorescence) produced in cells treated with 100 nM ghrelin for 0–60 min. c The ratio of SNAP production in cells treated with ghrelin or mock (medium) for 0–240 min, corresponding to ( b ). d Dynamics of G Luc production by cells treated with 100 nM ghrelin. The ratio of G Luc signals in the supernatants of cells treated with ghrelin or mock (medium) for indicated time. Note that over 90% of the newly produced G Luc is rapidly secreted from cells, thus minimising risks of intracellular protein degradation. e Signal-to-noise ratios calculated for the 15-min time point vs 0 time point (no ghrelin) of G Luc- and SNAP-based analysis. f RT-qPCR analysis of SNAP and G Luc mRNA levels in cells transfected with the corresponding plasmids and treated with mock or 100 nM ghrelin for 30 min ( N = 2). g Representative images (stacks of 7 focal planes) of Alexa Fluor 594 and EGFP fluorescence (showing nascent peptides (in red) and GHS-R1α (in blue), respectively) in mock and ghrelin-treated cells (100 nM ghrelin for 30 min); Click-iT ® HPG Alexa Fluor 594 Protein Synthesis Assay Kit was used. h Quantitative analysis of the Alexa Fluor 594 intensity signals in ghrelin- and mock-treated cells. i , j Distribution of ribosomes in sucrose density gradients and monosome/polysome (M/P) ratio in HEK293 GHS-R1α-EGFP+ cells treated with 100 nM ghrelin for 0–60 min ( N = 3). Error bars in ( c , d ) are presented for experiments repeated at least three times. In ( e , h ), boxes show median values and the interquartile range; in ( e ), whiskers extend to the minimal and maximal values; in ( h ), Altman-style whiskers extend to 5th and 95th percentiles, and all analysed cells are represented by data points ( n > 90 in each group, N = 3 independent experiments). Data are shown as individual data points (circles in f and j ; dots in h ), mean values (horizontal bars, f, h and j ) and mean ± SD. Asterisks show significant difference between ghrelin- and mock-treated (0-time point) cells. Statistical details: b, c Effect of ghrelin treatment time: SNAP (5, 15) = 27.048, p

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Produced, Quantitative RT-PCR

    Effects of GHS-R1α activation by ghrelin on the pathways regulating translation initiation and on the translation initiation complex in HEK293 GHS-R1α-EGFP+ cells. a , b Western blotting analysis of time- ( a ) and concentration- ( b ) dependent effects of ghrelin on the activity of AKT/mTOR and ERK signalling cascades, eIF2 phosphorylation and PDCD4 levels. In ( a ), 100 nM ghrelin was used; in ( b ), cells were treated for 30 min. c RPPA analysis of the effects of ghrelin treatment (500 nM, 1 h) on AMPK activity, reported by p-AMPKα (T172) and p-ACC (S79) levels ( N = 3). d Effect of ghrelin on GHS-R1α internalisation and cytosolic Ca 2+ levels: live cell confocal imaging analysis of translocation of GFP-tagged ghrelin receptor to cytosol and changes in Fura Red fluorescence upon ghrelin treatment. Note that a large proportion of GHS-R1α undergoes internalisation and aggregates in non-treated cells due to the constitutive activity of the receptor. Fura Red signals decrease upon Ca 2+ elevation; photo-bleaching of the dye is shown as mock. In all experiments ( N = 3), cells were pre-incubated in DMEM supplemented with 1% FBS for 12–14 h and then treated with 100 nM ghrelin. Fluorescence images are stacks of three (GFP) and five confocal planes (Fura Red) taken with 0.5 µm steps. e RPPA analysis of the effect of ghrelin treatment (500 nM, 1 h) on p-4E-BP1 (S65) levels; phosphorylation blocks the capacity of 4E-BP1 to inhibit initiation. f Composition of cap-bound proteins-regulators of translation initiation; changes in eIF4G and 4E-BP1 protein levels during 2 h treatment with ghrelin are shown. Input protein analysis is used as control; α-tubulin and eIF4G2 are detected only in the input samples, demonstrating specificity of cap binding. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks have not been used because of the complexity of plots; see statistical significance report below. Statistical details: a effect of ghrelin treatment time: (a) p-AKT (4,8) = 39.625, p
    Figure Legend Snippet: Effects of GHS-R1α activation by ghrelin on the pathways regulating translation initiation and on the translation initiation complex in HEK293 GHS-R1α-EGFP+ cells. a , b Western blotting analysis of time- ( a ) and concentration- ( b ) dependent effects of ghrelin on the activity of AKT/mTOR and ERK signalling cascades, eIF2 phosphorylation and PDCD4 levels. In ( a ), 100 nM ghrelin was used; in ( b ), cells were treated for 30 min. c RPPA analysis of the effects of ghrelin treatment (500 nM, 1 h) on AMPK activity, reported by p-AMPKα (T172) and p-ACC (S79) levels ( N = 3). d Effect of ghrelin on GHS-R1α internalisation and cytosolic Ca 2+ levels: live cell confocal imaging analysis of translocation of GFP-tagged ghrelin receptor to cytosol and changes in Fura Red fluorescence upon ghrelin treatment. Note that a large proportion of GHS-R1α undergoes internalisation and aggregates in non-treated cells due to the constitutive activity of the receptor. Fura Red signals decrease upon Ca 2+ elevation; photo-bleaching of the dye is shown as mock. In all experiments ( N = 3), cells were pre-incubated in DMEM supplemented with 1% FBS for 12–14 h and then treated with 100 nM ghrelin. Fluorescence images are stacks of three (GFP) and five confocal planes (Fura Red) taken with 0.5 µm steps. e RPPA analysis of the effect of ghrelin treatment (500 nM, 1 h) on p-4E-BP1 (S65) levels; phosphorylation blocks the capacity of 4E-BP1 to inhibit initiation. f Composition of cap-bound proteins-regulators of translation initiation; changes in eIF4G and 4E-BP1 protein levels during 2 h treatment with ghrelin are shown. Input protein analysis is used as control; α-tubulin and eIF4G2 are detected only in the input samples, demonstrating specificity of cap binding. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks have not been used because of the complexity of plots; see statistical significance report below. Statistical details: a effect of ghrelin treatment time: (a) p-AKT (4,8) = 39.625, p

    Techniques Used: Activation Assay, Western Blot, Concentration Assay, Activity Assay, Imaging, Translocation Assay, Fluorescence, Incubation, Binding Assay

    Proposed mechanism of eEF2K regulation upon GHS-R1α activation and analysis of pathways inhibiting eEF2K activity through Ser366 phosphorylation. a Schematics of eEF2K posttranslational modifications and ghrelin-triggered cascades modulating eEF2K activity and eEF2 phosphorylation state. Measured in this study parameters (using confocal microscopy, Western blotting and/or RPPA analysis of HEK293 GHS-R1α-EGFP+ cells) are in brackets, shown in red or green; italic is used for RPPA only data. Except for p-AMPKα (T172) and p-ACC (S79), which both demonstrated positive trends, and CREB (S133), which was shown in one replicate, all changes are statistically significant (one-way ANOVA and independent samples T test). The proposed pathways that de-suppress eEF2 upon ghrelin treatment are highlighted by a background yellow-grey arrow. b Time- and concentration-dependent effects of ghrelin treatment on p-eEF2K (S366) levels. c , d RPPA analysis of p-p70S6K (T389), p-p90RSK (T573), p-rpS6 (S240/S244) and p-rpS6 (S235/S236) levels. e Confocal microscopy analysis of p-rpS6 (S235/S236) levels; stacks of 6 fluorescence images taken with 0.5 µm step and single-plane DIC images show cellular p-rpS6 levels (Alexa Fluor 555) and nuclei (DAPI); 20 cells were analysed in three mock- and ghrelin-treated samples. Cells were pre-incubated for 15–16 h in serum-free DMEM supplemented with NEAA. In RPPA experiments, cells were treated with 500 nM ghrelin for 1 h, in confocal imaging experiments—with 100 nM ghrelin for 30 min. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin on p-eEF2K (S366): upper panel, effect of treatment time (4,20) = 16.730, p
    Figure Legend Snippet: Proposed mechanism of eEF2K regulation upon GHS-R1α activation and analysis of pathways inhibiting eEF2K activity through Ser366 phosphorylation. a Schematics of eEF2K posttranslational modifications and ghrelin-triggered cascades modulating eEF2K activity and eEF2 phosphorylation state. Measured in this study parameters (using confocal microscopy, Western blotting and/or RPPA analysis of HEK293 GHS-R1α-EGFP+ cells) are in brackets, shown in red or green; italic is used for RPPA only data. Except for p-AMPKα (T172) and p-ACC (S79), which both demonstrated positive trends, and CREB (S133), which was shown in one replicate, all changes are statistically significant (one-way ANOVA and independent samples T test). The proposed pathways that de-suppress eEF2 upon ghrelin treatment are highlighted by a background yellow-grey arrow. b Time- and concentration-dependent effects of ghrelin treatment on p-eEF2K (S366) levels. c , d RPPA analysis of p-p70S6K (T389), p-p90RSK (T573), p-rpS6 (S240/S244) and p-rpS6 (S235/S236) levels. e Confocal microscopy analysis of p-rpS6 (S235/S236) levels; stacks of 6 fluorescence images taken with 0.5 µm step and single-plane DIC images show cellular p-rpS6 levels (Alexa Fluor 555) and nuclei (DAPI); 20 cells were analysed in three mock- and ghrelin-treated samples. Cells were pre-incubated for 15–16 h in serum-free DMEM supplemented with NEAA. In RPPA experiments, cells were treated with 500 nM ghrelin for 1 h, in confocal imaging experiments—with 100 nM ghrelin for 30 min. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin on p-eEF2K (S366): upper panel, effect of treatment time (4,20) = 16.730, p

    Techniques Used: Activation Assay, Activity Assay, Confocal Microscopy, Western Blot, Concentration Assay, Fluorescence, Incubation, Imaging

    Effects of GHS-R1α activation by ghrelin on eEF2 phosphorylation. a Proposed contribution of elongation into ribosome turnover and protein production de novo. Decreased eEF2 phosphorylation is expected to increase elongation rate, ribosome availability and protein production levels. b , c Western blotting analysis of time- and concentration-dependent changes in eEF2 (Thr56) phosphorylation induced in HEK293 GHS-R1α-EGFP+ cells by ghrelin treatment; b 100 nM ghrelin for 15–60 min and c 4–100 nM ghrelin for 30 min. d Analysis of eEF2 phosphorylation in parental HEK293 cells lacking GHS-R1α. e Analysis of GHS-R1α protein expression in mouse embryonic hypothalamus cell lines E-N38 and E-N41; in E-N41, GHS-R1α protein levels are ~ 60% higher. f Ghrelin concentration-dependent decrease in p-eEF2 (Thr56) levels in E-N41 cells (30 min treatment). g Changes in the extracellular G Luc signals triggered by ghrelin treatment in E-N41 cells (200 nM, 30 min). Cells were pre-incubated for 14–16 h in serum-free DMEM supplemented with NEAA. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin treatment time: p-eEF2 (4,20) = 124.011, p
    Figure Legend Snippet: Effects of GHS-R1α activation by ghrelin on eEF2 phosphorylation. a Proposed contribution of elongation into ribosome turnover and protein production de novo. Decreased eEF2 phosphorylation is expected to increase elongation rate, ribosome availability and protein production levels. b , c Western blotting analysis of time- and concentration-dependent changes in eEF2 (Thr56) phosphorylation induced in HEK293 GHS-R1α-EGFP+ cells by ghrelin treatment; b 100 nM ghrelin for 15–60 min and c 4–100 nM ghrelin for 30 min. d Analysis of eEF2 phosphorylation in parental HEK293 cells lacking GHS-R1α. e Analysis of GHS-R1α protein expression in mouse embryonic hypothalamus cell lines E-N38 and E-N41; in E-N41, GHS-R1α protein levels are ~ 60% higher. f Ghrelin concentration-dependent decrease in p-eEF2 (Thr56) levels in E-N41 cells (30 min treatment). g Changes in the extracellular G Luc signals triggered by ghrelin treatment in E-N41 cells (200 nM, 30 min). Cells were pre-incubated for 14–16 h in serum-free DMEM supplemented with NEAA. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin treatment time: p-eEF2 (4,20) = 124.011, p

    Techniques Used: Activation Assay, Western Blot, Concentration Assay, Expressing, Incubation

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    Becton Dickinson anti ghrelin receptor ghsr extracellular antibody
    Roles of rpS6 kinases and PP2A in <t>ghrelin-induced</t> activation of eEF2 and protein synthesis. a The schematic of HEK293 GHS-R1α-EGFP+ cells treatment. b Changes in eEF2 and eEF2K phosphorylation induced by ghrelin after inhibition of either p90 RSK (10 µM BRD7389) or p70 S6K (1 µM PF4708671). c Effects of ghrelin on p-eEF2 (Thr56) and p-eEF2K (Ser366 and Ser500) levels in cells upon inhibition of p90 RSK and p70 S6K (double-treatment with BRD7389/PF4708671) or PP2A (OKA, 50 nM). In ( b , c ), cells were pre-treated with kinase or phosphatase inhibitors for 2 h prior to ghrelin addition (100 nM, 30 min). Changes in p-mTOR and p-ERK levels were assessed to confirm the GHS-R1α activation. d Changes in SNAP and G Luc production in cells treated with ghrelin upon inhibition of p70 S6K (1 µM PF4708671). Cells were pre-treated for 2 h and then treated with ghrelin (100 nM, 30 min) in the presence of PF4708671; an arrow shows the residual amounts of SNAP in cells lysed immediately after their treatment with SNAP block. e Comparative analysis of eEF2 response to ghrelin in resting and OKA-treated cells [extracted from ( c )]. The bottom panel shows that the pre-treatment with OKA causes an increase in phosphorylation of ERK, a common target of PP2A. Red rectangles ( b , c ) highlight effects of BRD7389 on total levels of mTOR, eEF2 and eEF2K proteins. Red asterisks indicate significant difference in the p-eEF2 (Thr56) response to ghrelin in mock-treated vs BRD- and PF-treated cells ( b ), or in mock-treated vs BRD/PF- and OKA-treated cells ( c ), red hash signs—significant difference in p-eEF2 response to ghrelin between BRD/PF- and OKA-treated cells. Black asterisks show the same for p-eEF2K (Ser366) levels. Columns/horizontal bars and gradient circles show mean values and individual data points, respectively. Statistical details: b effects of p90 RSK or p70 S6K inhibition on the ghrelin-induced changes in eEF2 and eEF2K phosphorylation; (a) p-eEF2 (2,6) = 29.442, p
    Anti Ghrelin Receptor Ghsr Extracellular Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ghrelin receptor ghsr extracellular antibody/product/Becton Dickinson
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ghrelin receptor ghsr extracellular antibody - by Bioz Stars, 2022-10
    86/100 stars
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    Alomone Labs antibody agr 031
    Roles of rpS6 kinases and PP2A in <t>ghrelin-induced</t> activation of eEF2 and protein synthesis. a The schematic of HEK293 GHS-R1α-EGFP+ cells treatment. b Changes in eEF2 and eEF2K phosphorylation induced by ghrelin after inhibition of either p90 RSK (10 µM BRD7389) or p70 S6K (1 µM PF4708671). c Effects of ghrelin on p-eEF2 (Thr56) and p-eEF2K (Ser366 and Ser500) levels in cells upon inhibition of p90 RSK and p70 S6K (double-treatment with BRD7389/PF4708671) or PP2A (OKA, 50 nM). In ( b , c ), cells were pre-treated with kinase or phosphatase inhibitors for 2 h prior to ghrelin addition (100 nM, 30 min). Changes in p-mTOR and p-ERK levels were assessed to confirm the GHS-R1α activation. d Changes in SNAP and G Luc production in cells treated with ghrelin upon inhibition of p70 S6K (1 µM PF4708671). Cells were pre-treated for 2 h and then treated with ghrelin (100 nM, 30 min) in the presence of PF4708671; an arrow shows the residual amounts of SNAP in cells lysed immediately after their treatment with SNAP block. e Comparative analysis of eEF2 response to ghrelin in resting and OKA-treated cells [extracted from ( c )]. The bottom panel shows that the pre-treatment with OKA causes an increase in phosphorylation of ERK, a common target of PP2A. Red rectangles ( b , c ) highlight effects of BRD7389 on total levels of mTOR, eEF2 and eEF2K proteins. Red asterisks indicate significant difference in the p-eEF2 (Thr56) response to ghrelin in mock-treated vs BRD- and PF-treated cells ( b ), or in mock-treated vs BRD/PF- and OKA-treated cells ( c ), red hash signs—significant difference in p-eEF2 response to ghrelin between BRD/PF- and OKA-treated cells. Black asterisks show the same for p-eEF2K (Ser366) levels. Columns/horizontal bars and gradient circles show mean values and individual data points, respectively. Statistical details: b effects of p90 RSK or p70 S6K inhibition on the ghrelin-induced changes in eEF2 and eEF2K phosphorylation; (a) p-eEF2 (2,6) = 29.442, p
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    Roles of rpS6 kinases and PP2A in ghrelin-induced activation of eEF2 and protein synthesis. a The schematic of HEK293 GHS-R1α-EGFP+ cells treatment. b Changes in eEF2 and eEF2K phosphorylation induced by ghrelin after inhibition of either p90 RSK (10 µM BRD7389) or p70 S6K (1 µM PF4708671). c Effects of ghrelin on p-eEF2 (Thr56) and p-eEF2K (Ser366 and Ser500) levels in cells upon inhibition of p90 RSK and p70 S6K (double-treatment with BRD7389/PF4708671) or PP2A (OKA, 50 nM). In ( b , c ), cells were pre-treated with kinase or phosphatase inhibitors for 2 h prior to ghrelin addition (100 nM, 30 min). Changes in p-mTOR and p-ERK levels were assessed to confirm the GHS-R1α activation. d Changes in SNAP and G Luc production in cells treated with ghrelin upon inhibition of p70 S6K (1 µM PF4708671). Cells were pre-treated for 2 h and then treated with ghrelin (100 nM, 30 min) in the presence of PF4708671; an arrow shows the residual amounts of SNAP in cells lysed immediately after their treatment with SNAP block. e Comparative analysis of eEF2 response to ghrelin in resting and OKA-treated cells [extracted from ( c )]. The bottom panel shows that the pre-treatment with OKA causes an increase in phosphorylation of ERK, a common target of PP2A. Red rectangles ( b , c ) highlight effects of BRD7389 on total levels of mTOR, eEF2 and eEF2K proteins. Red asterisks indicate significant difference in the p-eEF2 (Thr56) response to ghrelin in mock-treated vs BRD- and PF-treated cells ( b ), or in mock-treated vs BRD/PF- and OKA-treated cells ( c ), red hash signs—significant difference in p-eEF2 response to ghrelin between BRD/PF- and OKA-treated cells. Black asterisks show the same for p-eEF2K (Ser366) levels. Columns/horizontal bars and gradient circles show mean values and individual data points, respectively. Statistical details: b effects of p90 RSK or p70 S6K inhibition on the ghrelin-induced changes in eEF2 and eEF2K phosphorylation; (a) p-eEF2 (2,6) = 29.442, p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

    doi: 10.1007/s00018-022-04446-4

    Figure Lengend Snippet: Roles of rpS6 kinases and PP2A in ghrelin-induced activation of eEF2 and protein synthesis. a The schematic of HEK293 GHS-R1α-EGFP+ cells treatment. b Changes in eEF2 and eEF2K phosphorylation induced by ghrelin after inhibition of either p90 RSK (10 µM BRD7389) or p70 S6K (1 µM PF4708671). c Effects of ghrelin on p-eEF2 (Thr56) and p-eEF2K (Ser366 and Ser500) levels in cells upon inhibition of p90 RSK and p70 S6K (double-treatment with BRD7389/PF4708671) or PP2A (OKA, 50 nM). In ( b , c ), cells were pre-treated with kinase or phosphatase inhibitors for 2 h prior to ghrelin addition (100 nM, 30 min). Changes in p-mTOR and p-ERK levels were assessed to confirm the GHS-R1α activation. d Changes in SNAP and G Luc production in cells treated with ghrelin upon inhibition of p70 S6K (1 µM PF4708671). Cells were pre-treated for 2 h and then treated with ghrelin (100 nM, 30 min) in the presence of PF4708671; an arrow shows the residual amounts of SNAP in cells lysed immediately after their treatment with SNAP block. e Comparative analysis of eEF2 response to ghrelin in resting and OKA-treated cells [extracted from ( c )]. The bottom panel shows that the pre-treatment with OKA causes an increase in phosphorylation of ERK, a common target of PP2A. Red rectangles ( b , c ) highlight effects of BRD7389 on total levels of mTOR, eEF2 and eEF2K proteins. Red asterisks indicate significant difference in the p-eEF2 (Thr56) response to ghrelin in mock-treated vs BRD- and PF-treated cells ( b ), or in mock-treated vs BRD/PF- and OKA-treated cells ( c ), red hash signs—significant difference in p-eEF2 response to ghrelin between BRD/PF- and OKA-treated cells. Black asterisks show the same for p-eEF2K (Ser366) levels. Columns/horizontal bars and gradient circles show mean values and individual data points, respectively. Statistical details: b effects of p90 RSK or p70 S6K inhibition on the ghrelin-induced changes in eEF2 and eEF2K phosphorylation; (a) p-eEF2 (2,6) = 29.442, p

    Article Snippet: Antibodies were from: Alomone Labs, Israel (ghrelin receptor, № AGR-031); Becton Dickinson, NJ (eIF4G2 aka NAT1, № 610,742); Cell Signalling Technology, MA (CREB, № 1385; phospho-CREB (Ser133), № 9198; mTOR, № 2972; phospho-mTOR (Ser2448), № 2971; phospho-ERK1/2 (Thr202/Tyr204), № 9101; eEF2, № 2332; phospho-eEF2 (Thr56), № 2331; eEF2K, № 3692; phospho-eEF2K (Ser366), № 3691; eIF4G1, № 2498; 4E-BP1, № 9452; eIF2α, № 5324; phospho-eIF2α (Set51), № 3398; p-AKT (Ser473), № 4060; tuberin/TSC2, № 4304; phospho-TSC2 (Thr1462), № 3617; rpS6, №2217; phospho-rpS6 (Ser235/236), № 2271, PDCD4, № 9535); ECM Biosciences, KY (eEF2K Phospho-Regulation Antibody Sampler Kit, № EK6910, which includes antibodies against Ser78, Ser359, Thr348, Ser398, Ser500 in eEF2K and eEF2K C-terminus); Millipore, CA (ERK1/2, № 06–182); Proteintech, IL (AKT, № 10,176–2-AP); Sigma, MO (α-tubulin №T5168, HRP-conjugated anti-rabbit and anti-mouse antibodies, № A1949 and A0168); Thermo Fisher Scientific, MA (anti-rabbit Alexa Fluor 555-conjugated, № A-21428).

    Techniques: Activation Assay, Inhibition, Blocking Assay

    Susceptibility of ghrelin-induced de-suppression of eEF2 to metabolic stress. a The schematic of experiments and total ATP analysis in HEK293 GHS-R1α-EGFP+ cells supplied with or deprived of glucose for 15 h. b Western blotting analysis of changes in eEF2, eEF2K, mTOR and ERK phosphorylation induced by ghrelin treatment (50 nM and 100 nM, 30 min). c Glucose concentration-dependent changes of p-eEF2 and p-eEF2K levels in response to ghrelin (100 nM, 30 min), Western blotting analysis. The quantification curve shows dynamics of p-eEF2 levels, and include values corresponding to 1 mM glucose concentration (see Fig. S5a, c); maximal decrease in eEF2 phosphorylation corresponding to the ‘physiological’ range of glucose concentrations (2–10 mM) is highlighted by a rectangle. d Levels of eIF4G1 and 4E-BP1 proteins associated with the cap binding complex in resting and metabolically stressed cells stimulated with mock (DMEM) and 100 nM ghrelin for 30 min; in glucose-deprived cells the levels of the cap-bound initiation inhibitor 4E-BP1 strongly increase. e Effect of ghrelin (50 nM, 15 and 30 min) on protein phosphorylation in cells pre-treated for 2 h with FCCP (mitochondrial uncoupler, 1 µM) and quantitative analysis of p-eEF2 levels. Data are presented as mean ± SD. Gradient circles demonstrate individual data points. Significant difference from mock ( c , e ) and between glucose (+) and galactose (−) samples ( b ) is shown by asterisks. Statistical details: b effect of glucose deprivation on the ghrelin-induced changes in protein phosphorylation (50 nM ghrelin), N = 3, independent samples T test: p-mTOR t (4) = 2.109, p = 0.103; p-eEF2 t (4) = − 3.939, p = 0.017; p-eEF2K t (4) = 5.001, p = 0.007. c Effect of glucose concentration on the response to ghrelin: (a) p-eEF2 (4,8) = 28.243, p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

    doi: 10.1007/s00018-022-04446-4

    Figure Lengend Snippet: Susceptibility of ghrelin-induced de-suppression of eEF2 to metabolic stress. a The schematic of experiments and total ATP analysis in HEK293 GHS-R1α-EGFP+ cells supplied with or deprived of glucose for 15 h. b Western blotting analysis of changes in eEF2, eEF2K, mTOR and ERK phosphorylation induced by ghrelin treatment (50 nM and 100 nM, 30 min). c Glucose concentration-dependent changes of p-eEF2 and p-eEF2K levels in response to ghrelin (100 nM, 30 min), Western blotting analysis. The quantification curve shows dynamics of p-eEF2 levels, and include values corresponding to 1 mM glucose concentration (see Fig. S5a, c); maximal decrease in eEF2 phosphorylation corresponding to the ‘physiological’ range of glucose concentrations (2–10 mM) is highlighted by a rectangle. d Levels of eIF4G1 and 4E-BP1 proteins associated with the cap binding complex in resting and metabolically stressed cells stimulated with mock (DMEM) and 100 nM ghrelin for 30 min; in glucose-deprived cells the levels of the cap-bound initiation inhibitor 4E-BP1 strongly increase. e Effect of ghrelin (50 nM, 15 and 30 min) on protein phosphorylation in cells pre-treated for 2 h with FCCP (mitochondrial uncoupler, 1 µM) and quantitative analysis of p-eEF2 levels. Data are presented as mean ± SD. Gradient circles demonstrate individual data points. Significant difference from mock ( c , e ) and between glucose (+) and galactose (−) samples ( b ) is shown by asterisks. Statistical details: b effect of glucose deprivation on the ghrelin-induced changes in protein phosphorylation (50 nM ghrelin), N = 3, independent samples T test: p-mTOR t (4) = 2.109, p = 0.103; p-eEF2 t (4) = − 3.939, p = 0.017; p-eEF2K t (4) = 5.001, p = 0.007. c Effect of glucose concentration on the response to ghrelin: (a) p-eEF2 (4,8) = 28.243, p

    Article Snippet: Antibodies were from: Alomone Labs, Israel (ghrelin receptor, № AGR-031); Becton Dickinson, NJ (eIF4G2 aka NAT1, № 610,742); Cell Signalling Technology, MA (CREB, № 1385; phospho-CREB (Ser133), № 9198; mTOR, № 2972; phospho-mTOR (Ser2448), № 2971; phospho-ERK1/2 (Thr202/Tyr204), № 9101; eEF2, № 2332; phospho-eEF2 (Thr56), № 2331; eEF2K, № 3692; phospho-eEF2K (Ser366), № 3691; eIF4G1, № 2498; 4E-BP1, № 9452; eIF2α, № 5324; phospho-eIF2α (Set51), № 3398; p-AKT (Ser473), № 4060; tuberin/TSC2, № 4304; phospho-TSC2 (Thr1462), № 3617; rpS6, №2217; phospho-rpS6 (Ser235/236), № 2271, PDCD4, № 9535); ECM Biosciences, KY (eEF2K Phospho-Regulation Antibody Sampler Kit, № EK6910, which includes antibodies against Ser78, Ser359, Thr348, Ser398, Ser500 in eEF2K and eEF2K C-terminus); Millipore, CA (ERK1/2, № 06–182); Proteintech, IL (AKT, № 10,176–2-AP); Sigma, MO (α-tubulin №T5168, HRP-conjugated anti-rabbit and anti-mouse antibodies, № A1949 and A0168); Thermo Fisher Scientific, MA (anti-rabbit Alexa Fluor 555-conjugated, № A-21428).

    Techniques: Western Blot, Concentration Assay, Binding Assay, Metabolic Labelling

    Changes in protein synthesis in HEK293 GHS-R1α-EGFP+ cells in response to GHS-R1α activation by ghrelin. a Efficiency of cell transfection with a plasmid encoding SNAP ( > 55% SNAP-positive cells 24 h after the onset of transfection). b Representative fluorescent image of the gel showing the amounts of SNAP protein (647-SiR fluorescence) produced in cells treated with 100 nM ghrelin for 0–60 min. c The ratio of SNAP production in cells treated with ghrelin or mock (medium) for 0–240 min, corresponding to ( b ). d Dynamics of G Luc production by cells treated with 100 nM ghrelin. The ratio of G Luc signals in the supernatants of cells treated with ghrelin or mock (medium) for indicated time. Note that over 90% of the newly produced G Luc is rapidly secreted from cells, thus minimising risks of intracellular protein degradation. e Signal-to-noise ratios calculated for the 15-min time point vs 0 time point (no ghrelin) of G Luc- and SNAP-based analysis. f RT-qPCR analysis of SNAP and G Luc mRNA levels in cells transfected with the corresponding plasmids and treated with mock or 100 nM ghrelin for 30 min ( N = 2). g Representative images (stacks of 7 focal planes) of Alexa Fluor 594 and EGFP fluorescence (showing nascent peptides (in red) and GHS-R1α (in blue), respectively) in mock and ghrelin-treated cells (100 nM ghrelin for 30 min); Click-iT ® HPG Alexa Fluor 594 Protein Synthesis Assay Kit was used. h Quantitative analysis of the Alexa Fluor 594 intensity signals in ghrelin- and mock-treated cells. i , j Distribution of ribosomes in sucrose density gradients and monosome/polysome (M/P) ratio in HEK293 GHS-R1α-EGFP+ cells treated with 100 nM ghrelin for 0–60 min ( N = 3). Error bars in ( c , d ) are presented for experiments repeated at least three times. In ( e , h ), boxes show median values and the interquartile range; in ( e ), whiskers extend to the minimal and maximal values; in ( h ), Altman-style whiskers extend to 5th and 95th percentiles, and all analysed cells are represented by data points ( n > 90 in each group, N = 3 independent experiments). Data are shown as individual data points (circles in f and j ; dots in h ), mean values (horizontal bars, f, h and j ) and mean ± SD. Asterisks show significant difference between ghrelin- and mock-treated (0-time point) cells. Statistical details: b, c Effect of ghrelin treatment time: SNAP (5, 15) = 27.048, p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

    doi: 10.1007/s00018-022-04446-4

    Figure Lengend Snippet: Changes in protein synthesis in HEK293 GHS-R1α-EGFP+ cells in response to GHS-R1α activation by ghrelin. a Efficiency of cell transfection with a plasmid encoding SNAP ( > 55% SNAP-positive cells 24 h after the onset of transfection). b Representative fluorescent image of the gel showing the amounts of SNAP protein (647-SiR fluorescence) produced in cells treated with 100 nM ghrelin for 0–60 min. c The ratio of SNAP production in cells treated with ghrelin or mock (medium) for 0–240 min, corresponding to ( b ). d Dynamics of G Luc production by cells treated with 100 nM ghrelin. The ratio of G Luc signals in the supernatants of cells treated with ghrelin or mock (medium) for indicated time. Note that over 90% of the newly produced G Luc is rapidly secreted from cells, thus minimising risks of intracellular protein degradation. e Signal-to-noise ratios calculated for the 15-min time point vs 0 time point (no ghrelin) of G Luc- and SNAP-based analysis. f RT-qPCR analysis of SNAP and G Luc mRNA levels in cells transfected with the corresponding plasmids and treated with mock or 100 nM ghrelin for 30 min ( N = 2). g Representative images (stacks of 7 focal planes) of Alexa Fluor 594 and EGFP fluorescence (showing nascent peptides (in red) and GHS-R1α (in blue), respectively) in mock and ghrelin-treated cells (100 nM ghrelin for 30 min); Click-iT ® HPG Alexa Fluor 594 Protein Synthesis Assay Kit was used. h Quantitative analysis of the Alexa Fluor 594 intensity signals in ghrelin- and mock-treated cells. i , j Distribution of ribosomes in sucrose density gradients and monosome/polysome (M/P) ratio in HEK293 GHS-R1α-EGFP+ cells treated with 100 nM ghrelin for 0–60 min ( N = 3). Error bars in ( c , d ) are presented for experiments repeated at least three times. In ( e , h ), boxes show median values and the interquartile range; in ( e ), whiskers extend to the minimal and maximal values; in ( h ), Altman-style whiskers extend to 5th and 95th percentiles, and all analysed cells are represented by data points ( n > 90 in each group, N = 3 independent experiments). Data are shown as individual data points (circles in f and j ; dots in h ), mean values (horizontal bars, f, h and j ) and mean ± SD. Asterisks show significant difference between ghrelin- and mock-treated (0-time point) cells. Statistical details: b, c Effect of ghrelin treatment time: SNAP (5, 15) = 27.048, p

    Article Snippet: Antibodies were from: Alomone Labs, Israel (ghrelin receptor, № AGR-031); Becton Dickinson, NJ (eIF4G2 aka NAT1, № 610,742); Cell Signalling Technology, MA (CREB, № 1385; phospho-CREB (Ser133), № 9198; mTOR, № 2972; phospho-mTOR (Ser2448), № 2971; phospho-ERK1/2 (Thr202/Tyr204), № 9101; eEF2, № 2332; phospho-eEF2 (Thr56), № 2331; eEF2K, № 3692; phospho-eEF2K (Ser366), № 3691; eIF4G1, № 2498; 4E-BP1, № 9452; eIF2α, № 5324; phospho-eIF2α (Set51), № 3398; p-AKT (Ser473), № 4060; tuberin/TSC2, № 4304; phospho-TSC2 (Thr1462), № 3617; rpS6, №2217; phospho-rpS6 (Ser235/236), № 2271, PDCD4, № 9535); ECM Biosciences, KY (eEF2K Phospho-Regulation Antibody Sampler Kit, № EK6910, which includes antibodies against Ser78, Ser359, Thr348, Ser398, Ser500 in eEF2K and eEF2K C-terminus); Millipore, CA (ERK1/2, № 06–182); Proteintech, IL (AKT, № 10,176–2-AP); Sigma, MO (α-tubulin №T5168, HRP-conjugated anti-rabbit and anti-mouse antibodies, № A1949 and A0168); Thermo Fisher Scientific, MA (anti-rabbit Alexa Fluor 555-conjugated, № A-21428).

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Produced, Quantitative RT-PCR

    Effects of GHS-R1α activation by ghrelin on the pathways regulating translation initiation and on the translation initiation complex in HEK293 GHS-R1α-EGFP+ cells. a , b Western blotting analysis of time- ( a ) and concentration- ( b ) dependent effects of ghrelin on the activity of AKT/mTOR and ERK signalling cascades, eIF2 phosphorylation and PDCD4 levels. In ( a ), 100 nM ghrelin was used; in ( b ), cells were treated for 30 min. c RPPA analysis of the effects of ghrelin treatment (500 nM, 1 h) on AMPK activity, reported by p-AMPKα (T172) and p-ACC (S79) levels ( N = 3). d Effect of ghrelin on GHS-R1α internalisation and cytosolic Ca 2+ levels: live cell confocal imaging analysis of translocation of GFP-tagged ghrelin receptor to cytosol and changes in Fura Red fluorescence upon ghrelin treatment. Note that a large proportion of GHS-R1α undergoes internalisation and aggregates in non-treated cells due to the constitutive activity of the receptor. Fura Red signals decrease upon Ca 2+ elevation; photo-bleaching of the dye is shown as mock. In all experiments ( N = 3), cells were pre-incubated in DMEM supplemented with 1% FBS for 12–14 h and then treated with 100 nM ghrelin. Fluorescence images are stacks of three (GFP) and five confocal planes (Fura Red) taken with 0.5 µm steps. e RPPA analysis of the effect of ghrelin treatment (500 nM, 1 h) on p-4E-BP1 (S65) levels; phosphorylation blocks the capacity of 4E-BP1 to inhibit initiation. f Composition of cap-bound proteins-regulators of translation initiation; changes in eIF4G and 4E-BP1 protein levels during 2 h treatment with ghrelin are shown. Input protein analysis is used as control; α-tubulin and eIF4G2 are detected only in the input samples, demonstrating specificity of cap binding. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks have not been used because of the complexity of plots; see statistical significance report below. Statistical details: a effect of ghrelin treatment time: (a) p-AKT (4,8) = 39.625, p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

    doi: 10.1007/s00018-022-04446-4

    Figure Lengend Snippet: Effects of GHS-R1α activation by ghrelin on the pathways regulating translation initiation and on the translation initiation complex in HEK293 GHS-R1α-EGFP+ cells. a , b Western blotting analysis of time- ( a ) and concentration- ( b ) dependent effects of ghrelin on the activity of AKT/mTOR and ERK signalling cascades, eIF2 phosphorylation and PDCD4 levels. In ( a ), 100 nM ghrelin was used; in ( b ), cells were treated for 30 min. c RPPA analysis of the effects of ghrelin treatment (500 nM, 1 h) on AMPK activity, reported by p-AMPKα (T172) and p-ACC (S79) levels ( N = 3). d Effect of ghrelin on GHS-R1α internalisation and cytosolic Ca 2+ levels: live cell confocal imaging analysis of translocation of GFP-tagged ghrelin receptor to cytosol and changes in Fura Red fluorescence upon ghrelin treatment. Note that a large proportion of GHS-R1α undergoes internalisation and aggregates in non-treated cells due to the constitutive activity of the receptor. Fura Red signals decrease upon Ca 2+ elevation; photo-bleaching of the dye is shown as mock. In all experiments ( N = 3), cells were pre-incubated in DMEM supplemented with 1% FBS for 12–14 h and then treated with 100 nM ghrelin. Fluorescence images are stacks of three (GFP) and five confocal planes (Fura Red) taken with 0.5 µm steps. e RPPA analysis of the effect of ghrelin treatment (500 nM, 1 h) on p-4E-BP1 (S65) levels; phosphorylation blocks the capacity of 4E-BP1 to inhibit initiation. f Composition of cap-bound proteins-regulators of translation initiation; changes in eIF4G and 4E-BP1 protein levels during 2 h treatment with ghrelin are shown. Input protein analysis is used as control; α-tubulin and eIF4G2 are detected only in the input samples, demonstrating specificity of cap binding. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks have not been used because of the complexity of plots; see statistical significance report below. Statistical details: a effect of ghrelin treatment time: (a) p-AKT (4,8) = 39.625, p

    Article Snippet: Antibodies were from: Alomone Labs, Israel (ghrelin receptor, № AGR-031); Becton Dickinson, NJ (eIF4G2 aka NAT1, № 610,742); Cell Signalling Technology, MA (CREB, № 1385; phospho-CREB (Ser133), № 9198; mTOR, № 2972; phospho-mTOR (Ser2448), № 2971; phospho-ERK1/2 (Thr202/Tyr204), № 9101; eEF2, № 2332; phospho-eEF2 (Thr56), № 2331; eEF2K, № 3692; phospho-eEF2K (Ser366), № 3691; eIF4G1, № 2498; 4E-BP1, № 9452; eIF2α, № 5324; phospho-eIF2α (Set51), № 3398; p-AKT (Ser473), № 4060; tuberin/TSC2, № 4304; phospho-TSC2 (Thr1462), № 3617; rpS6, №2217; phospho-rpS6 (Ser235/236), № 2271, PDCD4, № 9535); ECM Biosciences, KY (eEF2K Phospho-Regulation Antibody Sampler Kit, № EK6910, which includes antibodies against Ser78, Ser359, Thr348, Ser398, Ser500 in eEF2K and eEF2K C-terminus); Millipore, CA (ERK1/2, № 06–182); Proteintech, IL (AKT, № 10,176–2-AP); Sigma, MO (α-tubulin №T5168, HRP-conjugated anti-rabbit and anti-mouse antibodies, № A1949 and A0168); Thermo Fisher Scientific, MA (anti-rabbit Alexa Fluor 555-conjugated, № A-21428).

    Techniques: Activation Assay, Western Blot, Concentration Assay, Activity Assay, Imaging, Translocation Assay, Fluorescence, Incubation, Binding Assay

    Proposed mechanism of eEF2K regulation upon GHS-R1α activation and analysis of pathways inhibiting eEF2K activity through Ser366 phosphorylation. a Schematics of eEF2K posttranslational modifications and ghrelin-triggered cascades modulating eEF2K activity and eEF2 phosphorylation state. Measured in this study parameters (using confocal microscopy, Western blotting and/or RPPA analysis of HEK293 GHS-R1α-EGFP+ cells) are in brackets, shown in red or green; italic is used for RPPA only data. Except for p-AMPKα (T172) and p-ACC (S79), which both demonstrated positive trends, and CREB (S133), which was shown in one replicate, all changes are statistically significant (one-way ANOVA and independent samples T test). The proposed pathways that de-suppress eEF2 upon ghrelin treatment are highlighted by a background yellow-grey arrow. b Time- and concentration-dependent effects of ghrelin treatment on p-eEF2K (S366) levels. c , d RPPA analysis of p-p70S6K (T389), p-p90RSK (T573), p-rpS6 (S240/S244) and p-rpS6 (S235/S236) levels. e Confocal microscopy analysis of p-rpS6 (S235/S236) levels; stacks of 6 fluorescence images taken with 0.5 µm step and single-plane DIC images show cellular p-rpS6 levels (Alexa Fluor 555) and nuclei (DAPI); 20 cells were analysed in three mock- and ghrelin-treated samples. Cells were pre-incubated for 15–16 h in serum-free DMEM supplemented with NEAA. In RPPA experiments, cells were treated with 500 nM ghrelin for 1 h, in confocal imaging experiments—with 100 nM ghrelin for 30 min. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin on p-eEF2K (S366): upper panel, effect of treatment time (4,20) = 16.730, p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

    doi: 10.1007/s00018-022-04446-4

    Figure Lengend Snippet: Proposed mechanism of eEF2K regulation upon GHS-R1α activation and analysis of pathways inhibiting eEF2K activity through Ser366 phosphorylation. a Schematics of eEF2K posttranslational modifications and ghrelin-triggered cascades modulating eEF2K activity and eEF2 phosphorylation state. Measured in this study parameters (using confocal microscopy, Western blotting and/or RPPA analysis of HEK293 GHS-R1α-EGFP+ cells) are in brackets, shown in red or green; italic is used for RPPA only data. Except for p-AMPKα (T172) and p-ACC (S79), which both demonstrated positive trends, and CREB (S133), which was shown in one replicate, all changes are statistically significant (one-way ANOVA and independent samples T test). The proposed pathways that de-suppress eEF2 upon ghrelin treatment are highlighted by a background yellow-grey arrow. b Time- and concentration-dependent effects of ghrelin treatment on p-eEF2K (S366) levels. c , d RPPA analysis of p-p70S6K (T389), p-p90RSK (T573), p-rpS6 (S240/S244) and p-rpS6 (S235/S236) levels. e Confocal microscopy analysis of p-rpS6 (S235/S236) levels; stacks of 6 fluorescence images taken with 0.5 µm step and single-plane DIC images show cellular p-rpS6 levels (Alexa Fluor 555) and nuclei (DAPI); 20 cells were analysed in three mock- and ghrelin-treated samples. Cells were pre-incubated for 15–16 h in serum-free DMEM supplemented with NEAA. In RPPA experiments, cells were treated with 500 nM ghrelin for 1 h, in confocal imaging experiments—with 100 nM ghrelin for 30 min. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin on p-eEF2K (S366): upper panel, effect of treatment time (4,20) = 16.730, p

    Article Snippet: Antibodies were from: Alomone Labs, Israel (ghrelin receptor, № AGR-031); Becton Dickinson, NJ (eIF4G2 aka NAT1, № 610,742); Cell Signalling Technology, MA (CREB, № 1385; phospho-CREB (Ser133), № 9198; mTOR, № 2972; phospho-mTOR (Ser2448), № 2971; phospho-ERK1/2 (Thr202/Tyr204), № 9101; eEF2, № 2332; phospho-eEF2 (Thr56), № 2331; eEF2K, № 3692; phospho-eEF2K (Ser366), № 3691; eIF4G1, № 2498; 4E-BP1, № 9452; eIF2α, № 5324; phospho-eIF2α (Set51), № 3398; p-AKT (Ser473), № 4060; tuberin/TSC2, № 4304; phospho-TSC2 (Thr1462), № 3617; rpS6, №2217; phospho-rpS6 (Ser235/236), № 2271, PDCD4, № 9535); ECM Biosciences, KY (eEF2K Phospho-Regulation Antibody Sampler Kit, № EK6910, which includes antibodies against Ser78, Ser359, Thr348, Ser398, Ser500 in eEF2K and eEF2K C-terminus); Millipore, CA (ERK1/2, № 06–182); Proteintech, IL (AKT, № 10,176–2-AP); Sigma, MO (α-tubulin №T5168, HRP-conjugated anti-rabbit and anti-mouse antibodies, № A1949 and A0168); Thermo Fisher Scientific, MA (anti-rabbit Alexa Fluor 555-conjugated, № A-21428).

    Techniques: Activation Assay, Activity Assay, Confocal Microscopy, Western Blot, Concentration Assay, Fluorescence, Incubation, Imaging

    Effects of GHS-R1α activation by ghrelin on eEF2 phosphorylation. a Proposed contribution of elongation into ribosome turnover and protein production de novo. Decreased eEF2 phosphorylation is expected to increase elongation rate, ribosome availability and protein production levels. b , c Western blotting analysis of time- and concentration-dependent changes in eEF2 (Thr56) phosphorylation induced in HEK293 GHS-R1α-EGFP+ cells by ghrelin treatment; b 100 nM ghrelin for 15–60 min and c 4–100 nM ghrelin for 30 min. d Analysis of eEF2 phosphorylation in parental HEK293 cells lacking GHS-R1α. e Analysis of GHS-R1α protein expression in mouse embryonic hypothalamus cell lines E-N38 and E-N41; in E-N41, GHS-R1α protein levels are ~ 60% higher. f Ghrelin concentration-dependent decrease in p-eEF2 (Thr56) levels in E-N41 cells (30 min treatment). g Changes in the extracellular G Luc signals triggered by ghrelin treatment in E-N41 cells (200 nM, 30 min). Cells were pre-incubated for 14–16 h in serum-free DMEM supplemented with NEAA. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin treatment time: p-eEF2 (4,20) = 124.011, p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis

    doi: 10.1007/s00018-022-04446-4

    Figure Lengend Snippet: Effects of GHS-R1α activation by ghrelin on eEF2 phosphorylation. a Proposed contribution of elongation into ribosome turnover and protein production de novo. Decreased eEF2 phosphorylation is expected to increase elongation rate, ribosome availability and protein production levels. b , c Western blotting analysis of time- and concentration-dependent changes in eEF2 (Thr56) phosphorylation induced in HEK293 GHS-R1α-EGFP+ cells by ghrelin treatment; b 100 nM ghrelin for 15–60 min and c 4–100 nM ghrelin for 30 min. d Analysis of eEF2 phosphorylation in parental HEK293 cells lacking GHS-R1α. e Analysis of GHS-R1α protein expression in mouse embryonic hypothalamus cell lines E-N38 and E-N41; in E-N41, GHS-R1α protein levels are ~ 60% higher. f Ghrelin concentration-dependent decrease in p-eEF2 (Thr56) levels in E-N41 cells (30 min treatment). g Changes in the extracellular G Luc signals triggered by ghrelin treatment in E-N41 cells (200 nM, 30 min). Cells were pre-incubated for 14–16 h in serum-free DMEM supplemented with NEAA. Data are shown as individual data points (gradient circles), mean values (horizontal bars) and mean ± SD. Asterisks indicate significant difference between non-treated (0 time point or 0 ghrelin concentration) and ghrelin-treated cells. Statistical details: b effect of ghrelin treatment time: p-eEF2 (4,20) = 124.011, p

    Article Snippet: Antibodies were from: Alomone Labs, Israel (ghrelin receptor, № AGR-031); Becton Dickinson, NJ (eIF4G2 aka NAT1, № 610,742); Cell Signalling Technology, MA (CREB, № 1385; phospho-CREB (Ser133), № 9198; mTOR, № 2972; phospho-mTOR (Ser2448), № 2971; phospho-ERK1/2 (Thr202/Tyr204), № 9101; eEF2, № 2332; phospho-eEF2 (Thr56), № 2331; eEF2K, № 3692; phospho-eEF2K (Ser366), № 3691; eIF4G1, № 2498; 4E-BP1, № 9452; eIF2α, № 5324; phospho-eIF2α (Set51), № 3398; p-AKT (Ser473), № 4060; tuberin/TSC2, № 4304; phospho-TSC2 (Thr1462), № 3617; rpS6, №2217; phospho-rpS6 (Ser235/236), № 2271, PDCD4, № 9535); ECM Biosciences, KY (eEF2K Phospho-Regulation Antibody Sampler Kit, № EK6910, which includes antibodies against Ser78, Ser359, Thr348, Ser398, Ser500 in eEF2K and eEF2K C-terminus); Millipore, CA (ERK1/2, № 06–182); Proteintech, IL (AKT, № 10,176–2-AP); Sigma, MO (α-tubulin №T5168, HRP-conjugated anti-rabbit and anti-mouse antibodies, № A1949 and A0168); Thermo Fisher Scientific, MA (anti-rabbit Alexa Fluor 555-conjugated, № A-21428).

    Techniques: Activation Assay, Western Blot, Concentration Assay, Expressing, Incubation