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    Novus Biologicals ago2 eif2c2 antibody
    Upregulation of KRas and loss of let-7a activity with neuronal differentiation. (A) Expression of KRas with increasing time of differentiation in PC12 cells was checked by Western blotting (WB) using β-actin as a loading control. (B) RT-PCR of undifferentiated and differentiated PC12 cells to check the Ras level using Rpl19 as a loading control. (C) (Top) Northern blotting detection of miRNA let-7a and U6 in naive PC12 cells and NGF-differentiated PC12 cells. U6 snRNA was used as a loading control. (Bottom) The intensities of the signals in the blots were quantified, and the relative level of let-7a was plotted. (D) Schematic representation of luciferase reporters showing the respective miRNA binding sites. (E) Fold repression of miRNA reporters with either one perfect let-7a miRNA binding site or multiple bulged let-7a miRNA binding sites in undifferentiated (Non-Diff.) and differentiated (Diff.) PC12 cells. The reporter RLHMGA2 contains the 3′ UTR of the HMGA2 gene, an endogenous let-7a target. RL mRNA with no let-7a binding sites in the 3′ UTR was used as a control. For the miR-122 activity assay, PC12 cells exogenously expressing miR-122 by transfection of phosphorylated miR-122 were used. (F) Decrease in let-7a activity in rat SCG primary neurons upon differentiation with NGF. The fold repression of let-7a miRNA reporters with multiple imperfect sites or one perfect let-7a site was measured and plotted. (G) Real-time PCR-based quantification to estimate the changes in the level of let-7a miRNA with differentiation of SCG primary neurons. U6 snRNA was used as a loading control. (H to K) Expression of <t>Ago2</t> in PC12 cells differentiated by NGF treatment (H, I) or in rat primary neurons (J, K) was checked. Western blotting-based (H, J) and RT-PCR-based (I, K) assays were used to estimate the expression of the neuronal differentiation marker gene GAP43 and the Ago2 protein in undifferentiated and differentiated PC12 cells and in rat primary neurons. β-Actin and Rpl19 were used as loading controls. The data are from four biological replicates and represent means ± SEMs. ns, nonsignificant; *, P < 0.05; **, P < 0.001; ***, P < 0.0001.
    Ago2 Eif2c2 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upregulation of KRas and loss of let-7a activity with neuronal differentiation. (A) Expression of KRas with increasing time of differentiation in PC12 cells was checked by Western blotting (WB) using β-actin as a loading control. (B) RT-PCR of undifferentiated and differentiated PC12 cells to check the Ras level using Rpl19 as a loading control. (C) (Top) Northern blotting detection of miRNA let-7a and U6 in naive PC12 cells and NGF-differentiated PC12 cells. U6 snRNA was used as a loading control. (Bottom) The intensities of the signals in the blots were quantified, and the relative level of let-7a was plotted. (D) Schematic representation of luciferase reporters showing the respective miRNA binding sites. (E) Fold repression of miRNA reporters with either one perfect let-7a miRNA binding site or multiple bulged let-7a miRNA binding sites in undifferentiated (Non-Diff.) and differentiated (Diff.) PC12 cells. The reporter RLHMGA2 contains the 3′ UTR of the HMGA2 gene, an endogenous let-7a target. RL mRNA with no let-7a binding sites in the 3′ UTR was used as a control. For the miR-122 activity assay, PC12 cells exogenously expressing miR-122 by transfection of phosphorylated miR-122 were used. (F) Decrease in let-7a activity in rat SCG primary neurons upon differentiation with NGF. The fold repression of let-7a miRNA reporters with multiple imperfect sites or one perfect let-7a site was measured and plotted. (G) Real-time PCR-based quantification to estimate the changes in the level of let-7a miRNA with differentiation of SCG primary neurons. U6 snRNA was used as a loading control. (H to K) Expression of Ago2 in PC12 cells differentiated by NGF treatment (H, I) or in rat primary neurons (J, K) was checked. Western blotting-based (H, J) and RT-PCR-based (I, K) assays were used to estimate the expression of the neuronal differentiation marker gene GAP43 and the Ago2 protein in undifferentiated and differentiated PC12 cells and in rat primary neurons. β-Actin and Rpl19 were used as loading controls. The data are from four biological replicates and represent means ± SEMs. ns, nonsignificant; *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

    doi: 10.1128/MCB.00054-16

    Figure Lengend Snippet: Upregulation of KRas and loss of let-7a activity with neuronal differentiation. (A) Expression of KRas with increasing time of differentiation in PC12 cells was checked by Western blotting (WB) using β-actin as a loading control. (B) RT-PCR of undifferentiated and differentiated PC12 cells to check the Ras level using Rpl19 as a loading control. (C) (Top) Northern blotting detection of miRNA let-7a and U6 in naive PC12 cells and NGF-differentiated PC12 cells. U6 snRNA was used as a loading control. (Bottom) The intensities of the signals in the blots were quantified, and the relative level of let-7a was plotted. (D) Schematic representation of luciferase reporters showing the respective miRNA binding sites. (E) Fold repression of miRNA reporters with either one perfect let-7a miRNA binding site or multiple bulged let-7a miRNA binding sites in undifferentiated (Non-Diff.) and differentiated (Diff.) PC12 cells. The reporter RLHMGA2 contains the 3′ UTR of the HMGA2 gene, an endogenous let-7a target. RL mRNA with no let-7a binding sites in the 3′ UTR was used as a control. For the miR-122 activity assay, PC12 cells exogenously expressing miR-122 by transfection of phosphorylated miR-122 were used. (F) Decrease in let-7a activity in rat SCG primary neurons upon differentiation with NGF. The fold repression of let-7a miRNA reporters with multiple imperfect sites or one perfect let-7a site was measured and plotted. (G) Real-time PCR-based quantification to estimate the changes in the level of let-7a miRNA with differentiation of SCG primary neurons. U6 snRNA was used as a loading control. (H to K) Expression of Ago2 in PC12 cells differentiated by NGF treatment (H, I) or in rat primary neurons (J, K) was checked. Western blotting-based (H, J) and RT-PCR-based (I, K) assays were used to estimate the expression of the neuronal differentiation marker gene GAP43 and the Ago2 protein in undifferentiated and differentiated PC12 cells and in rat primary neurons. β-Actin and Rpl19 were used as loading controls. The data are from four biological replicates and represent means ± SEMs. ns, nonsignificant; *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

    Article Snippet: Antibody against HRS was obtained from Bethyl; phosphotyrosine-specific antibody 4G10 and antibody against phosphoserine were obtained from Millipore; antibodies against HA, KRas, and β-actin were obtained from Roche; antibody against Alix and GAP43 were obtained from Santa Cruz; Ago2 (EIF2C2) antibody was obtained from Novus Biologicals; and antibodies against calnexin, LAMP1, MSK1, phospho-p38, phospho-mTOR, and phospho-MSK1 (p-MSK1) were obtained from Cell Signaling.

    Techniques: Activity Assay, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Luciferase, Binding Assay, Transfection, Real-time Polymerase Chain Reaction, Marker

    let-7a-mediated downregulation of KRas is necessary for differentiation of PC12 cells. (A) Effect of KRas downregulation on neuronal differentiation. PC12 cells were transfected with control siRNA and siRNA against KRas (siKRas), and the cells were allowed to differentiate. Western blotting was done to detect endogenous Ago2, the differentiation marker GAP43, and the signaling molecule KRas to check its downregulation. The phosphorylated forms of MSK1 and p38 were also checked. β-Actin was used as a loading control. (B) Effect of KRas knockdown on differentiation of PC12 cells. The lengths of neurites of green fluorescent protein-positive cells in a KRas-downregulated background were measured in the absence and presence of NGF in the medium. A similar measurement was done with control siRNA-transfected cells. (C and D) Differentiation status of cells treated with anti-let-7a or anti-miR-122 compared to that of undifferentiated and NGF-differentiated green fluorescent protein-positive cells (n = 20 cells). (C) Images of representative cells obtained by differential inference contrast. (D) Distribution of cells depending on their neurite length (n = 20 cells). (E) Effect of differentiation or anti-miRNA treatment on expression of KRas and the differentiation marker GAP43 determined by Western blotting. β-Actin was used as a loading control. (F and G) Effect of pre-let-7a expression on PC12 cells undergoing differentiation. PC12 cells grown in low-serum medium with NGF were cotransfected either with a control vector or with a pre-let-7a expression plasmid along with a green fluorescent protein expression vector. (F) Images of representative cells obtained by differential interference contrast (DIC). (G) The distribution of transfected cells (green fluorescent protein positive) on the basis of their neurite length in control or pre-let-7a-expressing PC12 cells (n = 20 cells). The data are from three biological replicates.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

    doi: 10.1128/MCB.00054-16

    Figure Lengend Snippet: let-7a-mediated downregulation of KRas is necessary for differentiation of PC12 cells. (A) Effect of KRas downregulation on neuronal differentiation. PC12 cells were transfected with control siRNA and siRNA against KRas (siKRas), and the cells were allowed to differentiate. Western blotting was done to detect endogenous Ago2, the differentiation marker GAP43, and the signaling molecule KRas to check its downregulation. The phosphorylated forms of MSK1 and p38 were also checked. β-Actin was used as a loading control. (B) Effect of KRas knockdown on differentiation of PC12 cells. The lengths of neurites of green fluorescent protein-positive cells in a KRas-downregulated background were measured in the absence and presence of NGF in the medium. A similar measurement was done with control siRNA-transfected cells. (C and D) Differentiation status of cells treated with anti-let-7a or anti-miR-122 compared to that of undifferentiated and NGF-differentiated green fluorescent protein-positive cells (n = 20 cells). (C) Images of representative cells obtained by differential inference contrast. (D) Distribution of cells depending on their neurite length (n = 20 cells). (E) Effect of differentiation or anti-miRNA treatment on expression of KRas and the differentiation marker GAP43 determined by Western blotting. β-Actin was used as a loading control. (F and G) Effect of pre-let-7a expression on PC12 cells undergoing differentiation. PC12 cells grown in low-serum medium with NGF were cotransfected either with a control vector or with a pre-let-7a expression plasmid along with a green fluorescent protein expression vector. (F) Images of representative cells obtained by differential interference contrast (DIC). (G) The distribution of transfected cells (green fluorescent protein positive) on the basis of their neurite length in control or pre-let-7a-expressing PC12 cells (n = 20 cells). The data are from three biological replicates.

    Article Snippet: Antibody against HRS was obtained from Bethyl; phosphotyrosine-specific antibody 4G10 and antibody against phosphoserine were obtained from Millipore; antibodies against HA, KRas, and β-actin were obtained from Roche; antibody against Alix and GAP43 were obtained from Santa Cruz; Ago2 (EIF2C2) antibody was obtained from Novus Biologicals; and antibodies against calnexin, LAMP1, MSK1, phospho-p38, phospho-mTOR, and phospho-MSK1 (p-MSK1) were obtained from Cell Signaling.

    Techniques: Transfection, Western Blot, Marker, Expressing, Plasmid Preparation

    The loss of miRNA binding to Ago2 during differentiation of sympathetic neurons causes a reduction of miRNA activity in differentiated cells. (A) Loss of miRNAs from immunoprecipitated Ago2 isolated from NGF-differentiated PC12 cells. The relative levels of the miRNAs in total and Ago2-immunoprecipitated RNAs were estimated by real-time RT-PCR, and Ago2 was detected by Western blot analysis. (B) Increased phosphorylation of Ago2 in differentiating PC12 cells. (Left) The phosphorylated form of Ago2 (p-Ago2) was detected with a phosphotyrosine-specific antibody (4G10) in affinity-purified Ago2 materials isolated from naive and differentiated PC12 cell extracts. (Right) Relative levels of phosphorylated Ago2 in naive and differentiated PC12 cells. (C) Western blotting was done to determine the Ago2 expression level in the sympathetic and cortical regions of newborn and 7-day-old rat pup brains. (D) (Top) Western blots indicate the input and HA-immunoprecipitated levels of NHA-LacZ (control), NHA-Ago2, and NHA-Ago3 (fusion proteins with viral N peptide and hemagglutinin [HA] epitopes at their N termini). (Bottom) NHA-Ago2- and NHA-Ago3-associated let-7a miRNA levels, along with NHA-LacZ-associated let-7a miRNA levels as a control, in PC12 cells. (E) Levels of miR-128a (left) and let-7a (right) in total RNA isolated from the SCG and cortical regions of rat brain were estimated. (F) Relative amounts of miR-128a (top) and let-7a (middle) associated with endogenous Ago2 in the sympathetic and cortical regions of newborn and 7-day-old rat pup brains. (Bottom) Western blotting indicates the amount of immunoprecipitated Ago2 or its phosphorylated form in the sympathetic and cortical regions of newborn and 7-day-old rat pup brains. The data are from three biological replicates for PC12 cells and four biological replicates for rat pups. The relative level of phosphorylated Ago2 is shown. Data represent means ± SEMs. ns, nonsignificant; *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

    doi: 10.1128/MCB.00054-16

    Figure Lengend Snippet: The loss of miRNA binding to Ago2 during differentiation of sympathetic neurons causes a reduction of miRNA activity in differentiated cells. (A) Loss of miRNAs from immunoprecipitated Ago2 isolated from NGF-differentiated PC12 cells. The relative levels of the miRNAs in total and Ago2-immunoprecipitated RNAs were estimated by real-time RT-PCR, and Ago2 was detected by Western blot analysis. (B) Increased phosphorylation of Ago2 in differentiating PC12 cells. (Left) The phosphorylated form of Ago2 (p-Ago2) was detected with a phosphotyrosine-specific antibody (4G10) in affinity-purified Ago2 materials isolated from naive and differentiated PC12 cell extracts. (Right) Relative levels of phosphorylated Ago2 in naive and differentiated PC12 cells. (C) Western blotting was done to determine the Ago2 expression level in the sympathetic and cortical regions of newborn and 7-day-old rat pup brains. (D) (Top) Western blots indicate the input and HA-immunoprecipitated levels of NHA-LacZ (control), NHA-Ago2, and NHA-Ago3 (fusion proteins with viral N peptide and hemagglutinin [HA] epitopes at their N termini). (Bottom) NHA-Ago2- and NHA-Ago3-associated let-7a miRNA levels, along with NHA-LacZ-associated let-7a miRNA levels as a control, in PC12 cells. (E) Levels of miR-128a (left) and let-7a (right) in total RNA isolated from the SCG and cortical regions of rat brain were estimated. (F) Relative amounts of miR-128a (top) and let-7a (middle) associated with endogenous Ago2 in the sympathetic and cortical regions of newborn and 7-day-old rat pup brains. (Bottom) Western blotting indicates the amount of immunoprecipitated Ago2 or its phosphorylated form in the sympathetic and cortical regions of newborn and 7-day-old rat pup brains. The data are from three biological replicates for PC12 cells and four biological replicates for rat pups. The relative level of phosphorylated Ago2 is shown. Data represent means ± SEMs. ns, nonsignificant; *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

    Article Snippet: Antibody against HRS was obtained from Bethyl; phosphotyrosine-specific antibody 4G10 and antibody against phosphoserine were obtained from Millipore; antibodies against HA, KRas, and β-actin were obtained from Roche; antibody against Alix and GAP43 were obtained from Santa Cruz; Ago2 (EIF2C2) antibody was obtained from Novus Biologicals; and antibodies against calnexin, LAMP1, MSK1, phospho-p38, phospho-mTOR, and phospho-MSK1 (p-MSK1) were obtained from Cell Signaling.

    Techniques: Binding Assay, Activity Assay, Immunoprecipitation, Isolation, Quantitative RT-PCR, Western Blot, Affinity Purification, Expressing

    Phosphorylation of Ago2 is necessary for differentiation of PC12 cells. (A) Images of PC12 cells transfected with FLAG-HA (FHA)-tagged versions of Ago2 (wild type) and two different mutants, Ago2 Y529E (phosphor mimetic) and Ago2 Y529F (phosphor dead), obtained by microscopy. Transfected cells were detected by HA (green) expression, and cells were costained with Rck/p54 (red). Merged images and images obtained by differential interference contrast (DIC) are given. (B) Lengths of neurites of transfected PC12 cells. (C) Scheme of the experiment showing incubation of FLAG-tagged Ago2 purified from HEK293 cells with the lysates of differentiated PC12 cells. (Left) The end product, indicating the degradation of unphosphorylated Ago2 and the stability of the phosphorylated form of Ago2. (Right) In vitro kinase assay with FLAG-HA-Ago2 and lysates obtained from undifferentiated and differentiated PC12 cells, along with a buffer control, to check the phosphorylated Ago2 (P-Ago2) level after a reaction detected by phospho-Tyr-specific 4G10 antibody. (D) Cell fractionation assay to detect the distribution of let-7a miRNA and Ago2 along with various organellar markers. Alix and HRS (MVB/endosome markers), LAMP1 (a lysosomal marker), and calnexin (an endoplasmic reticulum marker) were used as marker proteins. (E and F) Lighter fractions (fractions 2, 3, and 4) and heavier fractions (fractions 7, 8, and 9) of the OptiPrep density gradient were fractionated and were pooled separately, and endogenous Ago2 was immunoprecipitated to check the Tyr phosphorylation level using phosphotyrosine-specific 4G10 antibody (E) and the associated let-7a level (F). The data are from four biological replicates and represent means ± SEMs. *, P < 0.05; ***, P < 0.0001.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

    doi: 10.1128/MCB.00054-16

    Figure Lengend Snippet: Phosphorylation of Ago2 is necessary for differentiation of PC12 cells. (A) Images of PC12 cells transfected with FLAG-HA (FHA)-tagged versions of Ago2 (wild type) and two different mutants, Ago2 Y529E (phosphor mimetic) and Ago2 Y529F (phosphor dead), obtained by microscopy. Transfected cells were detected by HA (green) expression, and cells were costained with Rck/p54 (red). Merged images and images obtained by differential interference contrast (DIC) are given. (B) Lengths of neurites of transfected PC12 cells. (C) Scheme of the experiment showing incubation of FLAG-tagged Ago2 purified from HEK293 cells with the lysates of differentiated PC12 cells. (Left) The end product, indicating the degradation of unphosphorylated Ago2 and the stability of the phosphorylated form of Ago2. (Right) In vitro kinase assay with FLAG-HA-Ago2 and lysates obtained from undifferentiated and differentiated PC12 cells, along with a buffer control, to check the phosphorylated Ago2 (P-Ago2) level after a reaction detected by phospho-Tyr-specific 4G10 antibody. (D) Cell fractionation assay to detect the distribution of let-7a miRNA and Ago2 along with various organellar markers. Alix and HRS (MVB/endosome markers), LAMP1 (a lysosomal marker), and calnexin (an endoplasmic reticulum marker) were used as marker proteins. (E and F) Lighter fractions (fractions 2, 3, and 4) and heavier fractions (fractions 7, 8, and 9) of the OptiPrep density gradient were fractionated and were pooled separately, and endogenous Ago2 was immunoprecipitated to check the Tyr phosphorylation level using phosphotyrosine-specific 4G10 antibody (E) and the associated let-7a level (F). The data are from four biological replicates and represent means ± SEMs. *, P < 0.05; ***, P < 0.0001.

    Article Snippet: Antibody against HRS was obtained from Bethyl; phosphotyrosine-specific antibody 4G10 and antibody against phosphoserine were obtained from Millipore; antibodies against HA, KRas, and β-actin were obtained from Roche; antibody against Alix and GAP43 were obtained from Santa Cruz; Ago2 (EIF2C2) antibody was obtained from Novus Biologicals; and antibodies against calnexin, LAMP1, MSK1, phospho-p38, phospho-mTOR, and phospho-MSK1 (p-MSK1) were obtained from Cell Signaling.

    Techniques: Transfection, Microscopy, Expressing, Incubation, Purification, In Vitro, Kinase Assay, Cell Fractionation, Marker, Immunoprecipitation

    Neuronal differentiation activates the p38 MAPK pathway for the phosphorylation of Ago2 and inactivation of miRNPs. (A) Expression levels of phospho-mTOR (p-mTOR), phospho-p38 (p-p38), and phospho-MSK1 (p-MSK1) in undifferentiated and differentiated PC12 cells and in rat primary neurons. β-Actin was used as a loading control. (B) Effect of 16 h of treatment of differentiated PC12 cells with 1 μM SB203580 (SB), an inhibitor of the p38 signaling pathway, on the expression of Ago2, GAP43, phospho-mTOR, phospho-MSK1, and phospho-p38. β-Actin was used as a loading control. (C) Real-time PCR-based quantification of let-7a miRNA levels in undifferentiated and differentiated PC12 cells without and with application of 1 μM p38 inhibitor for 16 h. (D to F) In sets of experiments similar to those described for panel B, a luciferase assay was done to check the effect of the p38 inhibitor on let-7a activity (D), endogenous Ago2 was immunoprecipitated and 4G10 levels were checked (E, top) and quantified (E, bottom), and real-time PCR was performed to measure the relative level of immunoprecipitated Ago2-associated let-7a miRNA in PC12 cells (F). (G) Distribution of the lengths of neurites of green fluorescent protein (GFP)-positive PC12 cells upon treatment as described in the legend to panel B without and with application of the p38 inhibitor SB203580 in the absence or presence of NGF in the medium. DMSO, dimethyl sulfoxide. The data are from four biological replicates and represent means ± SEMs. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

    doi: 10.1128/MCB.00054-16

    Figure Lengend Snippet: Neuronal differentiation activates the p38 MAPK pathway for the phosphorylation of Ago2 and inactivation of miRNPs. (A) Expression levels of phospho-mTOR (p-mTOR), phospho-p38 (p-p38), and phospho-MSK1 (p-MSK1) in undifferentiated and differentiated PC12 cells and in rat primary neurons. β-Actin was used as a loading control. (B) Effect of 16 h of treatment of differentiated PC12 cells with 1 μM SB203580 (SB), an inhibitor of the p38 signaling pathway, on the expression of Ago2, GAP43, phospho-mTOR, phospho-MSK1, and phospho-p38. β-Actin was used as a loading control. (C) Real-time PCR-based quantification of let-7a miRNA levels in undifferentiated and differentiated PC12 cells without and with application of 1 μM p38 inhibitor for 16 h. (D to F) In sets of experiments similar to those described for panel B, a luciferase assay was done to check the effect of the p38 inhibitor on let-7a activity (D), endogenous Ago2 was immunoprecipitated and 4G10 levels were checked (E, top) and quantified (E, bottom), and real-time PCR was performed to measure the relative level of immunoprecipitated Ago2-associated let-7a miRNA in PC12 cells (F). (G) Distribution of the lengths of neurites of green fluorescent protein (GFP)-positive PC12 cells upon treatment as described in the legend to panel B without and with application of the p38 inhibitor SB203580 in the absence or presence of NGF in the medium. DMSO, dimethyl sulfoxide. The data are from four biological replicates and represent means ± SEMs. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

    Article Snippet: Antibody against HRS was obtained from Bethyl; phosphotyrosine-specific antibody 4G10 and antibody against phosphoserine were obtained from Millipore; antibodies against HA, KRas, and β-actin were obtained from Roche; antibody against Alix and GAP43 were obtained from Santa Cruz; Ago2 (EIF2C2) antibody was obtained from Novus Biologicals; and antibodies against calnexin, LAMP1, MSK1, phospho-p38, phospho-mTOR, and phospho-MSK1 (p-MSK1) were obtained from Cell Signaling.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Immunoprecipitation

    Involvement of p38 downstream factor MSK1 on Ago phosphorylation upon neuronal differentiation. (A) Immunoprecipitation of endogenous Ago2 from PC12 cells followed by detection of phospho-mTOR, phospho-p38, and phospho-MSK1 to find their association with Ago2 in naive and differentiated PC12 cells. Immunoprecipitation of endogenous p-MSK1 from PC12 cells followed by detection of endogenous Ago2 in naive and differentiated PC12 cells was used to document the changes in the association of p-MSK1 with Ago2 upon differentiation. (B) Immunoprecipitation of Ago2 in differentiated PC12 cells without and with application of the p38 inhibitor to detect endogenous Ago2, its phosphorylated form (as detected by the phosphotyrosine-specific 4G10 antibody), and the associated MSK1 and phospho-MSK1 by Western blotting. Input samples denote the change in the levels of MSK1 and p-MSK1 without and with the use of the p38 inhibitor SB203580. (C) Lengths of neurites of green fluorescent protein-positive PC12 cells treated with control siRNA (siCon) and siRNA against MSK1 in the absence and presence of NGF in the medium (n = 15 cells). (D) Western blotting to detect the effect of MSK1 knockdown by siMSK1 transfection compared to the effect of control siRNA on differentiated PC12 cells to detect changes in the levels of endogenous Ago2 and the differentiation marker GAP43. Immunoprecipitation of endogenous Ago2 from the lysates of siMSK1- and control siRNA-transfected PC12 cells was done to check the levels of tyrosine and serine phosphorylation of Ago2 by using a phosphotyrosine-specific antibody (4G10) and phosphoserine-specific antibody (P-Ser). (E) The relative level of let-7 associated with immunoprecipitated Ago2. Values were normalized to the amount of immunoprecipitated Ago2. (F) A luciferase assay was done to check the effect of siRNA-mediated MSK1 knockdown on let-7a activity compared to that of control siRNA in PC12 cells. (G) Immunoprecipitation with phosphoserine antibody to detect changes in the amount of Ser-phosphorylated Ago2 protein in naive and differentiated PC12 cells. On immunoprecipitation with phosphoserine antibody, two bands for Ago2 were observed. The lower band of 95 kDa was that of Ago2 (reconfirmed with Ago2-specific antibody) and is marked with an arrow. (H) Immunoprecipitation of Ago2 from undifferentiated and differentiated cells was done, and phosphoserine levels were detected with a specific antibody. (I) In vitro kinase assay with p-MSK1 isolated from PC12 cells. Lysates from undifferentiated and differentiated PC12 cells were immunoprecipitated with p-MSK1 antibody, the immunoprecipitate was incubated with an equal amount of immunologically isolated FLAG-HA-tagged Ago2 from HEK293 cells, and a kinase assay was performed. The blots indicate the change in the levels of tyrosine and serine phosphorylation of FLAG-HA-tagged Ago2 in naive and differentiated PC12 cells. The data are from four biological replicates and represent means ± SEMs. *, P < 0.05; **, P < 0.001.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

    doi: 10.1128/MCB.00054-16

    Figure Lengend Snippet: Involvement of p38 downstream factor MSK1 on Ago phosphorylation upon neuronal differentiation. (A) Immunoprecipitation of endogenous Ago2 from PC12 cells followed by detection of phospho-mTOR, phospho-p38, and phospho-MSK1 to find their association with Ago2 in naive and differentiated PC12 cells. Immunoprecipitation of endogenous p-MSK1 from PC12 cells followed by detection of endogenous Ago2 in naive and differentiated PC12 cells was used to document the changes in the association of p-MSK1 with Ago2 upon differentiation. (B) Immunoprecipitation of Ago2 in differentiated PC12 cells without and with application of the p38 inhibitor to detect endogenous Ago2, its phosphorylated form (as detected by the phosphotyrosine-specific 4G10 antibody), and the associated MSK1 and phospho-MSK1 by Western blotting. Input samples denote the change in the levels of MSK1 and p-MSK1 without and with the use of the p38 inhibitor SB203580. (C) Lengths of neurites of green fluorescent protein-positive PC12 cells treated with control siRNA (siCon) and siRNA against MSK1 in the absence and presence of NGF in the medium (n = 15 cells). (D) Western blotting to detect the effect of MSK1 knockdown by siMSK1 transfection compared to the effect of control siRNA on differentiated PC12 cells to detect changes in the levels of endogenous Ago2 and the differentiation marker GAP43. Immunoprecipitation of endogenous Ago2 from the lysates of siMSK1- and control siRNA-transfected PC12 cells was done to check the levels of tyrosine and serine phosphorylation of Ago2 by using a phosphotyrosine-specific antibody (4G10) and phosphoserine-specific antibody (P-Ser). (E) The relative level of let-7 associated with immunoprecipitated Ago2. Values were normalized to the amount of immunoprecipitated Ago2. (F) A luciferase assay was done to check the effect of siRNA-mediated MSK1 knockdown on let-7a activity compared to that of control siRNA in PC12 cells. (G) Immunoprecipitation with phosphoserine antibody to detect changes in the amount of Ser-phosphorylated Ago2 protein in naive and differentiated PC12 cells. On immunoprecipitation with phosphoserine antibody, two bands for Ago2 were observed. The lower band of 95 kDa was that of Ago2 (reconfirmed with Ago2-specific antibody) and is marked with an arrow. (H) Immunoprecipitation of Ago2 from undifferentiated and differentiated cells was done, and phosphoserine levels were detected with a specific antibody. (I) In vitro kinase assay with p-MSK1 isolated from PC12 cells. Lysates from undifferentiated and differentiated PC12 cells were immunoprecipitated with p-MSK1 antibody, the immunoprecipitate was incubated with an equal amount of immunologically isolated FLAG-HA-tagged Ago2 from HEK293 cells, and a kinase assay was performed. The blots indicate the change in the levels of tyrosine and serine phosphorylation of FLAG-HA-tagged Ago2 in naive and differentiated PC12 cells. The data are from four biological replicates and represent means ± SEMs. *, P < 0.05; **, P < 0.001.

    Article Snippet: Antibody against HRS was obtained from Bethyl; phosphotyrosine-specific antibody 4G10 and antibody against phosphoserine were obtained from Millipore; antibodies against HA, KRas, and β-actin were obtained from Roche; antibody against Alix and GAP43 were obtained from Santa Cruz; Ago2 (EIF2C2) antibody was obtained from Novus Biologicals; and antibodies against calnexin, LAMP1, MSK1, phospho-p38, phospho-mTOR, and phospho-MSK1 (p-MSK1) were obtained from Cell Signaling.

    Techniques: Immunoprecipitation, Western Blot, Transfection, Marker, Luciferase, Activity Assay, In Vitro, Kinase Assay, Isolation, Incubation

    Mechanism of let-7a deactivation and its coupling with differentiation of neuronal cells. KRas is known to be involved in the PC12 differentiation pathway. KRas is a let-7a-regulated mRNA. Uncoupling of let-7a from Ago2 causes a loss of activity, and the KRas level increases with increasing time of differentiation. The uncoupling of miRNA from Ago2 is caused by its phosphorylation via the p38 MAPK/p-MSK1 pathway. Thus, Ago2 gets phosphorylated via a p-MSK1-driven pathway and let-7a activity decreases, leading to the upregulation of KRas, which ensures the continuous activity of the p38 MAPK pathway and the differentiation of sympathetic neurons.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

    doi: 10.1128/MCB.00054-16

    Figure Lengend Snippet: Mechanism of let-7a deactivation and its coupling with differentiation of neuronal cells. KRas is known to be involved in the PC12 differentiation pathway. KRas is a let-7a-regulated mRNA. Uncoupling of let-7a from Ago2 causes a loss of activity, and the KRas level increases with increasing time of differentiation. The uncoupling of miRNA from Ago2 is caused by its phosphorylation via the p38 MAPK/p-MSK1 pathway. Thus, Ago2 gets phosphorylated via a p-MSK1-driven pathway and let-7a activity decreases, leading to the upregulation of KRas, which ensures the continuous activity of the p38 MAPK pathway and the differentiation of sympathetic neurons.

    Article Snippet: Antibody against HRS was obtained from Bethyl; phosphotyrosine-specific antibody 4G10 and antibody against phosphoserine were obtained from Millipore; antibodies against HA, KRas, and β-actin were obtained from Roche; antibody against Alix and GAP43 were obtained from Santa Cruz; Ago2 (EIF2C2) antibody was obtained from Novus Biologicals; and antibodies against calnexin, LAMP1, MSK1, phospho-p38, phospho-mTOR, and phospho-MSK1 (p-MSK1) were obtained from Cell Signaling.

    Techniques: Activity Assay