Journal: Biomolecules

Article Title: Study of Protein Phosphatase 2A (PP2A) Activity in LPS-Induced Tolerance Using Fluorescence-Based and Immunoprecipitation-Aided Methodology

doi: 10.3390/biom5031284

Figure Lengend Snippet: Immunoprecipitation-aided fluorescence method of measuring PP2A activity in tolerized peritoneal macrophages. Anti-PP2A IgG was incubated with Protein A/G magnetic beads for 30 min and then cross-linked to the beads by DSS. Cell lysates were then incubated with antibody-conjugated beads for 1 hr at room temperature and the beads were removed of were washed to remove non-specific binding. Assay buffer containing DiFMUP and NiCl 2 was added to the beads, mixed and incubated at room temperature for 30 min. Supernatant removed of beads was measured on a SpectraMax M3 plate reader. ( A ) Anti-PP2A dose-response. Three doses of anti-PP2A IgG were cross-linked to the beads and incubated with the same amount of input lysates (75 µg) for the phosphatase assay; ( B ) Flow through section after beads were removed was examined for the presence of PP2A to determine pulldown efficiency; ( C ) Input cell lysates dose-response. Same amount of anti-PP2A IgG (2.5 µg) was incubated with two doses of cell lysates (75 µg and 150 µg). PP2A activity was measured by DiFMU method. Y axis shows the absolute values of DiFMU in solutions; ( D ) LPS tolerance induced upregulated PP2A-activity in peritoneal macrophages. 75 µg of cell lysates harvested from naïve or tolerant peritoneal macrophages were subjected to immunoprecipitation with 2.5 µg of anti-PP2AC antibody. Y-axis shows the absolute values of DiFMU.

Article Snippet: Normal mouse IgG or anti-PP2A was incubated with Pierce Protein A/G Magnetic beads at room temperature for 15–30 min and then the beads were washed twice to remove unbound antibodies.

Techniques: Immunoprecipitation, Fluorescence, Activity Assay, Incubation, Magnetic Beads, Binding Assay, Phosphatase Assay

Journal: Molecular Brain

Article Title: Acute 5-HT7 receptor activation increases NMDA-evoked currents and differentially alters NMDA receptor subunit phosphorylation and trafficking in hippocampal neurons

doi: 10.1186/1756-6606-6-24

Figure Lengend Snippet: LP 12 application results in the selective reduction of the surface expression of NR2B. A ) CA1 hippocampal slices were treated with vehicle, 300 nM LP 12, or LP 12 + 1 μM SB 258719 for 5 min. The slices were biotinylated and lysates were incubated overnight with streptavidin beads. Data represent the average and standard error of 6 independent experiments. * p < 0.05 LP 12 vs. vehicle ANOVA analysis with Dunnett’s post-test. B ) Total lysates or biotinylated samples were blotted using antibodies against NR2B or β-actin.

Article Snippet: Lysate protein concentrations were normalized and lysates were incubated with streptavidin beads overnight at 4°C (Sigma).

Techniques: Expressing, Incubation