rabbit antiactive extracellular signal-regulated protein kinase (erk)  (Promega)

Journal: Endocrinology

Article Title: Glucocorticoids Induce Rapid Up-Regulation of Mitogen-Activated Protein Kinase Phosphatase-1 and Dephosphorylation of Extracellular Signal-Regulated Kinase and Impair Proliferation in Human and Mouse Osteoblast Cell Lines

doi:

Figure Lengend Snippet: MKP-1 protein is markedly up-regulated by Dex treatment and this correlates precisely with dephosphorylation of ERK. Human MG-63 (A) and mouse MBA-15.4 (B) osteoblast cell lines were subjected to 1 μM Dex treatment for 1–8 h while growing rapidly in 10% FCS. Because MKP-1 is rapidly degraded following synthesis and action, cells were pretreated for 1 h with the 26S proteasome inhibitor, MG132 (30 μM) where indicated. MKP-1 protein was quantified by Western blot and densitometry. MKP-1 up-regulation in human MG-63 cells (C) and concomitant ERK dephosphorylation (D) by 1 μM Dex treatment for 4 or 7 h was compared following treatment for 5 min with 100 ng/ml TPA and 2 h with 20% FCS, and also in normally growing MG-63 osteoblasts in 10% serum. Results shown are representative of at least two experimental repeats. P-ERK quantifications in (D) were performed by stripping and reprobing the same blot with antiactive ERK antibody, and densitometric values are normalized for total ERK content.

Article Snippet: Polyclonal antiactive ERK (threonine and tyrosine phosphorylated, p42 and p44) was from New England Biolabs, Inc. (Beverly, MA).

Techniques: De-Phosphorylation Assay, Western Blot, Stripping Membranes

Journal: Developmental biology

Article Title: Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

doi: 10.1016/j.ydbio.2005.06.016

Figure Lengend Snippet: Fig. 3. Inhibition of phosphatases by okadaic acid allows CPEB phosphory- lation and cyclin B synthesis in enucleated oocyte. Normal (controls) or enucleated A. aranciacus oocytes were injected (+) or not () with 1 AM okadaic acid (OA) and treated with 1-Ma for the indicated time (min). The upper panel shows an anti-CPEB Western blot (CPEB) and the lower the autoradiography of 35S-pulse labeling, for 10 min, in a similar experiment (35S). The migration position of cyclin B is indicated by an arrow.

Article Snippet: Rabbit polyclonal antibodies against full-length M. glacialis cyclin B (Offner et al., 2003) were used for immunoprecipitation of cdc2-cyclin B. Antiactive-ERK antibodies were from Santa Cruz Biotechnology (sc-7383).

Techniques: Inhibition, Injection, Western Blot, Autoradiography, Labeling, Migration

Journal: Developmental biology

Article Title: Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

doi: 10.1016/j.ydbio.2005.06.016

Figure Lengend Snippet: Fig. 4. Microinjection of Inh-2 in enucleated oocytes restores cyclin B translation, CPEB phosphorylation and Aurora activation. (A) A. aranciacus oocytes were enucleated (E) or enucleated and injected with 15 AM Inh-2 (E + I-2) or untreated (ctrl). Then 1-MA was added for 70 min before 35S-pulse labeling, for 10 min. Batches of 10 oocytes were submitted to SDS–PAGE and autoradiography. The migration position of cyclin B is indicated by an arrow. (B) Western blot with anti-CPEB of normal (controls) or enucleated A. aranciacus oocytes injected (+) or not () with 15 AM Inh-2 and treated with 1-MA for the indicated time (minutes). (C) MBP-kinase activity of anti-Aurora immunoprecipitates from normal or enucleated, microinjected with 15 AM Inh-2 (E + I-2) or not (E), A. aranciacus oocytes taken at the indicated time after 1-MA addition. The autoradiography shows the radioactivity incorporated in MBP after in vitro phosphorylation with 32P-ATP and electrophoresis.

Article Snippet: Rabbit polyclonal antibodies against full-length M. glacialis cyclin B (Offner et al., 2003) were used for immunoprecipitation of cdc2-cyclin B. Antiactive-ERK antibodies were from Santa Cruz Biotechnology (sc-7383).

Techniques: Microinjection, Phospho-proteomics, Activation Assay, Injection, Labeling, SDS Page, Autoradiography, Migration, Western Blot, Activity Assay, Radioactivity, In Vitro, Electrophoresis

Journal: Developmental biology

Article Title: Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

doi: 10.1016/j.ydbio.2005.06.016

Figure Lengend Snippet: Fig. 6. CPEB is hyperphosphorylated, in vitro, by cdc2 but not by Aurora. (A) Autoradiography of 35S-labeled CPEB incubated for 2 h with an immunoprecipitate, from M-phase M. glacialis extract, of non-immune serum (C), anti-Aurora (Aur) and anti-cyclin B (CB). The migration position of unmodified CPEB is indicated by an arrow and that of the slowest migrating form by an arrowhead. (B) In a similar experiment, CPEB incubated with anti-cyclin B immunoprecipitate was treated (+) or not () with lambda phosphatase. (C) Similar experiment in which CPEB was incubated with anti-cyclin B immunoprecipitate for 0, 20, 60 or 120 min.

Article Snippet: Rabbit polyclonal antibodies against full-length M. glacialis cyclin B (Offner et al., 2003) were used for immunoprecipitation of cdc2-cyclin B. Antiactive-ERK antibodies were from Santa Cruz Biotechnology (sc-7383).

Techniques: In Vitro, Autoradiography, Labeling, Incubation, Migration

Journal: Developmental biology

Article Title: Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

doi: 10.1016/j.ydbio.2005.06.016

Figure Lengend Snippet: Fig. 5. Inhibitor-2 activates bacterially produced but not endogenous Aurora, and activated Aurora does not induce CPEB phosphorylation, when microinjected in enucleated oocytes. (A) MBP-kinase activity of bacterially produced starfish Aurora treated (+) or not () with Inh-2 (8 AM for 10 min at 20-C), either directly (direct) or after immunoprecipitation with anti- Aurora (IP). The autoradiography shows the radioactivity incorporated in MBP after in vitro phosphorylation with 32P-ATPand electrophoresis. (B) MBP-kinase activity of anti-Aurora immunoprecipitates from extracts of M. glacialis oocytes blocked in prophase (G2) or in M-phase (M) and treated (+) or not () with Inh-2 (8 AM for 10 min at 20-C). (C) MBP-kinase activity of bacterially produced starfish Aurora incubated (+) or not () with purified cdc2-cyclin B from M-phase M. glacialis extract, and adenosine thiotriphosphate. The autoradiography shows the radioactivity incorporated in MBP after in vitro phosphorylation with 32P-ATP and electrophoresis. (D) Western blot with anti-CPEB of enucleated A. auranciacus oocytes injected (en+ Aur) or not (en) with thiophosphorylated Aurora before a 60-min treatment with 1-MA, or normal oocytes (ctrl) treated for 50 min with 1-MA.

Article Snippet: Rabbit polyclonal antibodies against full-length M. glacialis cyclin B (Offner et al., 2003) were used for immunoprecipitation of cdc2-cyclin B. Antiactive-ERK antibodies were from Santa Cruz Biotechnology (sc-7383).

Techniques: Produced, Phospho-proteomics, Activity Assay, Immunoprecipitation, Autoradiography, Radioactivity, In Vitro, Electrophoresis, Incubation, Purification, Western Blot, Injection

Journal: Developmental biology

Article Title: Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

doi: 10.1016/j.ydbio.2005.06.016

Figure Lengend Snippet: Fig. 8. CPEB degradation is not required for cyclin B translation. (A) Only cyclin B translation readily occurs after completion of meiotic maturation. Autoradiography of 35S-pulse labeling, for 10 min, of A. aranciacus oocytes taken at the indicated time after 1-MA addition. The migration position of cyclin B is indicated by an arrow. The occurrences of germinal vesicle breakdown (GVBD), polar bodies emission (pb1, pb2) and of the pronucleus stage (PN) are indicated. (B) Western blot with anti-CPEB of normal (controls) or enucleated oocytes injected with Inh-2 (E + I-2) and treated with 1-Ma for the indicated time (min).

Article Snippet: Rabbit polyclonal antibodies against full-length M. glacialis cyclin B (Offner et al., 2003) were used for immunoprecipitation of cdc2-cyclin B. Antiactive-ERK antibodies were from Santa Cruz Biotechnology (sc-7383).

Techniques: Autoradiography, Labeling, Migration, Western Blot, Injection