nonimmunized mouse igg serum  (Vector Laboratories)

Journal: The Journal of biological chemistry

Article Title: Regulation of estrogen receptor alpha-mediated transcription by a direct interaction with protein phosphatase 2A.

doi: 10.1074/jbc.M210949200

Figure Lengend Snippet: FIG. 1. The phosphatase inhibitor okadaic acid increases phosphoryla- tion of ER. A, ER immunopellets were prepared from Rad91 cells cultured in 32P-containing serum-free medium (SFM) for 2 h in the presence or absence of 10 nM E2 or 1 M OA. OA increased phosphoryl- ation of the ER. B, ER immunopellets or nonimmune IgG pellets (NI) prepared from Rad91 cells following a 2-h exposure to 1 M OA or vehicle were used in an in vitro kinase reaction. OA increased phos- phorylation of a predominant band that migrated at the expected size for GFP- ER, and immunoblot analysis identified this band as ER. C, Rad91 cells were transfected with either wild-type ER (WT) or a point mutant containing an al- anine substitution for serine 118 (S118A). ER immunopellets were obtained from cells treated with 1 M OA or vehicle- treated cells, and an in vitro kinase reac- tion was performed. Autoradiography demonstrated that OA enhanced the phosphorylation of the wild-type ER, but not the S118A mutant. Serine 118 was also identified as the OA-induced phos- phorylation site in wild-type ER by immu- noblotting with an antibody specific for the 118 phospho-form of the ER (118-P).

Article Snippet: Immunoprecipitation—Cells grown until 90% confluence were harvested in lysis buffer (20 mM Tris-Cl, pH 7.5, 0.137 M NaCl, 2 mm EDTA, pH 7.4, 1% Triton, 10% glycerol, 25 mM -glycerol phosphate, and phenylmethylsulfonyl fluoride and protease inhibitor mixture), and the lysates were incubated overnight at 4 °C with 5 g of nonimmune mouse IgG, mouse monoclonal anti-ER antibody (Ab7; Neo Markers, Freemont, CA), nonimmune goat serum IgG, or goat polyclonal antiPP2A antibody (G-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Techniques: Cell Culture, In Vitro, Western Blot, Transfection, Mutagenesis, Autoradiography, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Regulation of estrogen receptor alpha-mediated transcription by a direct interaction with protein phosphatase 2A.

doi: 10.1074/jbc.M210949200

Figure Lengend Snippet: FIG. 2. ER associates with and is a substrate for PP2A. A, phosphatase assays were performed using 32P-myelin basic protein as a substrate in the presence of nonimmune IgG, or ER immunopellets derived from BAEC, human aortic endothelial cells (EAhy926 cells), and Rad91 cells infected with adeno-GFP-ER. Left panel, significant phosphatase activity was detected in ER immunopellets derived from all three cell types. Right panel, the phosphatase activity associated with the Rad91 cell ER immunopellet was partially inhibited by 10 nM OA, and more completely inhibited by 1 M okadaic acid. Bars represent the mean S.E. of three independent experiments. *, p 0.05 versus no okadaic acid. B, protein lysates were immunoprecipitated with an anti-ER antibody (IP-ER), or with nonimmune IgG (NI) and the resulting immunopellets were resolved by SDS-PAGE and immunoblotted for PP2A, PP1, and ER. Both PP2A and PP1 were detected in ER immunopellets, but not in nonimmune IgG pellets derived from BAEC. PP2A and PP1 were also detected in ER immunopellets, but not in nonimmune IgG pellets, and derived Rad91 cells were infected with adeno-GFP-ER (ER). No phosphatases were detected in immunopellets prepared from Rad91 cells infected with the control virus adeno-GFP (ER). C, ER immunopellets from GFP-ER-infected, okadaic acid-treated Rad91 cells were phosphorylated in vitro and the reaction products were then used as substrate in phosphatase reactions. Autoradiography demonstrated diminished labeling of ER following incubation with 0.1–0.2 units of highly purified PP2A (pPP2A), and this was blocked by co-incubation with 10 nM OA.

Article Snippet: Immunoprecipitation—Cells grown until 90% confluence were harvested in lysis buffer (20 mM Tris-Cl, pH 7.5, 0.137 M NaCl, 2 mm EDTA, pH 7.4, 1% Triton, 10% glycerol, 25 mM -glycerol phosphate, and phenylmethylsulfonyl fluoride and protease inhibitor mixture), and the lysates were incubated overnight at 4 °C with 5 g of nonimmune mouse IgG, mouse monoclonal anti-ER antibody (Ab7; Neo Markers, Freemont, CA), nonimmune goat serum IgG, or goat polyclonal antiPP2A antibody (G-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Techniques: Derivative Assay, Infection, Activity Assay, Immunoprecipitation, SDS Page, Control, Virus, In Vitro, Autoradiography, Labeling, Incubation, Purification