Journal: The Journal of biological chemistry

Article Title: Mapping the Protein Phosphatase-2B Anchoring Site on AKAP79

doi: 10.1074/jbc.M207833200

Figure Lengend Snippet: COS7 cells were transfected with myc-PP2B catalytic A subunit and a variety of anchoring protein constructs. A–C, cells were fixed and immunostained for Myc-PP2B (anti-Myc, Texas Red). D–F, double immunofluorescence staining of cells to detect myc-PP2B (red) and AKAP79 (green). G–I, double immunofluorescence detection of myc-PP2B (red) and AKAP79-GFP (green). J–L, double immunofluorescence detection of myc-PP2B (red) and AKAP18-GFP (green). Analysis was performed on an inverted microscope with a digital CCD camera. Overlap of the green and red signals is seen as yellow in the composite images.

Article Snippet: The washed coverslips were mounted on glass slides and analyzed using a either a Zeiss Axiovert TV-135 inverted microscope (63×plan-apo, oil, 1.4 NA) equipped with a digital CCD camera (Sensys) and Meta-GFP imaging software (Universal Imaging) or a Nikon TE-300 inverted microscope (100×plan-apo, oil, 1.4 NA) equipped with a digital CCD camera (Princeton Instruments) and Slidebook 3.0 imaging software (Intelligent Imaging Innovations).

Techniques: Transfection, Construct, Double Immunofluorescence Staining, Immunofluorescence, Inverted Microscopy

Journal: The Journal of biological chemistry

Article Title: Mapping the Protein Phosphatase-2B Anchoring Site on AKAP79

doi: 10.1074/jbc.M207833200

Figure Lengend Snippet: A, schematic diagram of the AKAP79 fragments used to analyze PP2B localization in COS7 cells. The first and last residue of each construct and the positions of internal deletions are indicated. GFP was fused at the C terminus of each fragment. The approximate location of the PKA (blue) and PKC (cyan)-binding sites and subcellular targeting domains (green) are indicated. Constructs capable of targeting PP2B (filled boxes) are indicated. B–S, COS7 cells transfected with myc-PP2B catalytic subunit and a variety of anchoring protein constructs. Cells were fixed, immunostained, and imaged to visualize the location of each AKAP79 fragment (green) and Myc-PP2B (red). Analysis was performed on an inverted microscope with a digital CCD camera. Overlap of the green and red signals is seen as yellow in the composite images. B–D, characterization of the AKAP79-(1–360) fragment. E–G, characterization of the AKAP79-(1–315) fragment. H–J, characterization of the AKAP79-(1–108) fragment. K–M, characterization of the AKAP79-(Δ151–315) fragment. N–P, characterization of the AKAP79-(Δ151–360) fragment. Q–S, characterization of the AKAP79-(Δ315–360) fragment.

Article Snippet: The washed coverslips were mounted on glass slides and analyzed using a either a Zeiss Axiovert TV-135 inverted microscope (63×plan-apo, oil, 1.4 NA) equipped with a digital CCD camera (Sensys) and Meta-GFP imaging software (Universal Imaging) or a Nikon TE-300 inverted microscope (100×plan-apo, oil, 1.4 NA) equipped with a digital CCD camera (Princeton Instruments) and Slidebook 3.0 imaging software (Intelligent Imaging Innovations).

Techniques: Construct, Binding Assay, Transfection, Inverted Microscopy

Journal: The Journal of biological chemistry

Article Title: Mapping the Protein Phosphatase-2B Anchoring Site on AKAP79

doi: 10.1074/jbc.M207833200

Figure Lengend Snippet: A, purified AKAP79 and PP2B holoenzymes were incubated in vitro with increasing concentrations (0–10 μM) of 330–357 peptide or unrelated control peptide (10 μM, Ht31 PKA-R-binding peptide). PP2B binding to AKAP79 was then analyzed by immunoprecipitation (IP) with anti-AKAP79. Top panel, co-purification of PP2B in the presence of increasing AKAP79-(330–357) peptide (indicated above each lane) was detected by immunoblotting with polyclonal antibodies against the phosphatase. The migration position of the A subunit of PP2B is indicated. Bottom panel, equal levels of AKAP79 in each experiment were confirmed by immunoblot. The migration position of AKAP79 is indicated. B, immunoblots from four independent experiments were analyzed densitometrically using NIH image software for quantification. Values on the y axis are normalized (set at 1.0) to the amount of PP2B immunoprecipitated with AKAP79 in the absence of peptide. Normalized intensity values are represented as the mean ± S.D. across all experiments. The concentration of the AKAP79-(330–357) peptide is indicated below each column. C, schematic diagram depicting the region of AKAP79 used to generate a soluble GFP fusion protein for cellular expression in COS7 cells. The first and last residues of the anchoring protein fragment are indicated. D–K, cellular expression of the AKAP79-(321–360)-GFP fusion protein is sufficient to disrupt PP2B/AKAP interaction inside cells. COS7 cells were transfected with control plasmid or the AKAP79-(321–360)-GFP construct. Both samples were also transfected with vectors encoding full-length AKAP79 and the A subunit of PP2B. Transfected cells were then fixed, stained, and analyzed by triple-fluorescence microscopy for GFP (green) (D and H), AKAP79-Cy5 (blue) (E and I), and PP2B-Texas Red (red) (F and J). G and K, an overlay of all three fluorescence channels is shown in the composite panel.

Article Snippet: The washed coverslips were mounted on glass slides and analyzed using a either a Zeiss Axiovert TV-135 inverted microscope (63×plan-apo, oil, 1.4 NA) equipped with a digital CCD camera (Sensys) and Meta-GFP imaging software (Universal Imaging) or a Nikon TE-300 inverted microscope (100×plan-apo, oil, 1.4 NA) equipped with a digital CCD camera (Princeton Instruments) and Slidebook 3.0 imaging software (Intelligent Imaging Innovations).

Techniques: Purification, Incubation, In Vitro, Binding Assay, Immunoprecipitation, Copurification, Western Blot, Migration, Software, Concentration Assay, Expressing, Transfection, Plasmid Preparation, Construct, Staining, Fluorescence, Microscopy