Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Immunoblot analysis of RI, RII, and AKAP100 in adult hearts. Soluble fraction of the rat left ventricle extracts (lanes 1 and 2 , from a preparative gel) and crude protein extracts from isolated rat left ventricular myocytes (lane 3 , 50 μg/lane) were separated on 10% SDS-PAGE gels by using the mini gel system (Bio-Rad Laboratories, Hercules, CA). The immunoblots were incubated with RI-specific monoclonal antibody (lane 1 ; 0.25 μg/ml), RII-specific goat antibodies (lane 2 ; 10 μg/ml) and AKAP100-specific rabbit antibodies (lane 3 ; 3.5 μg/ml), respectively. The protein bands were visualized by color development as described in the text (lanes 1 and 2 ) or by ECL (lane 3 ). Molecular weight markers are in kD.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Western Blot, Isolation, SDS Page, Incubation, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Immunoblot, immunoprecipitation, and cAMP-agarose copurification analyses of AKAP100 in the rat heart. In A , human (lane 2 ) and rat (lane 3 ) left ventricle homogenates were separated on a 10% SDS-PAGE gel (100 μg/lane) followed by immunoblotting with anti-AKAP100 antibodies. Unstained molecular weight markers were loaded on lane 1 . In B , rat left ventricle homogenates (0.5 ml) were incubated with 10 μg of AKAP100-specific rabbit IgGs (lane 1 ), 10 μg of RII-specific goat IgGs (lane 2 ), and 200 μl of cAMP-agarose (lane 3 ). The AKAP100-IgG complexes were precipitated by incubation with Protein G Sepharose beads. The precipitated proteins were solubilized from the agarose or sepharose beads with SDS-PAGE sample buffer followed by immunoblotting with anti-AKAP100 antibodies. As controls for the assay, 2 μg of rabbit IgGs and goat IgGs were loaded on lanes 4 and 5 , respectively. Prestained molecular weight markers are loaded on lane 6 . In C , human (lane 1 ) and rat (lane 2 ) left ventricle homogenates and human AKAP100 recombinant bacterial lysates (lane 3 ) were separated on a 10% SDS-PAGE gel (100 μg/lane) followed by immunoblotting with anti-AKAP100 antibodies that have been previously preabsorbed by AKAP100 recombinant bacterial lysates. As a control, 2 μg of rabbit IgGs were loaded on lane 4 . In D , AKAP100 recombinant bacterial lysates (50 μg/lane) were separated on a 10% SDS-PAGE gel followed by immunoblotting with affinity-purified anti-AKAP100 IgGs as described in the text. The Hoefer ( A–C ) and Bio-Rad ( D ) SDS-PAGE systems were used for these studies. A and B represent different lanes from the same gel. The protein bands in A–C were visualized by color development, whereas the immunoblot shown in D was developed by ECL. Molecular weight markers are in kD.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Western Blot, Immunoprecipitation, Copurification, SDS Page, Molecular Weight, Incubation, Recombinant, Control, Affinity Purification

Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Characterization of human AKAP100 and RII recombinant proteins. In A , human AKAP100 recombinant bacterial lysates were analyzed by immunblotting (lanes 1 and 2 , from a preparative gel) and RII overlay (lanes 3 and 4 , 100 μg/lane) assays. Lanes 1 and 2 were incubated with anti-6 His tag antibody and anti-AKAP100 antibodies, respectively. The protein bands were visualized by ECL. The RII overlay assay was performed as described in the text with (lane 4 ) or without (lane 3 ) the inhibitory peptide Ht31. The 32 P-labeled RII binding protein was detected by PhosphorImaging (Molecular Dynamics, Inc.). In B , purified RII recombinant protein was analyzed by Coomassie blue staining (lane 1 , 50 μg/lane) and immunoblotting (lane 2 , 10 μg/lane) with anti-RII antibodies. The protein band was visualized by ECL. Lane 3 was loaded with 10 μl of 32 P-labeled RII, which was detected by PhosphorImaging. Molecular weights are in kD.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Recombinant, Incubation, Overlay Assay, Labeling, Binding Assay, Purification, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Immunolocalization of AKAP100 in longitudinal sections of the rat papillary muscle. Longitudinal sections of rat papillary muscles were immunostained with AKAP100-specific rabbit antibodies and examined by confocal microscopy. Localization of the primary antibodies was detected with LRSC-conjugated donkey anti–rabbit IgG antibodies. The transverse periodic staining ( T ), the nuclear staining ( N ), and the intercalated disc staining ( I ) are indicated. Arrowheads label the AKAP100 staining between the transverse staining. Bar, 5 μm.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Muscles, Confocal Microscopy, Staining

Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Colocalization of AKAP100 with α-actinin and RyR in longitudinal sections of cardiac myocytes. Longitudinal cryostat sections of rat papillary muscles ( A–C ) or freshly isolated adult rat left ventricular myocytes ( D–F ) were double-immunostained with either α-actinin–specific ( A ) and AKAP100-specific ( B ) antibodies or RyR-specific ( D ) and AKAP100-specific ( E ) antibodies. The anti-AKAP100 rabbit antibodies were detected with LRSC-conjugated donkey anti–rabbit IgG antibodies. Localization of the RyR- and α-actinin-specific monoclonal antibodies were visualized by using FITC-conjugated donkey anti–mouse IgG antibodies. C is the superimposed image of A and B ; F combines from the images in D and E . Transverse periodic staining ( T ), sarcolemmal staining ( S ), nuclear staining ( N ), and intercalated disc staining ( I ) are indicated. In C , arrowheads indicate the longitudinal AKAP100 staining localized between the transverse staining, whereas in D and F , arrowheads label the punctate RyR staining. The nucleus without staining is labeled with an N . Bar, 5 μm.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Muscles, Isolation, Bioprocessing, Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Colocalization of RII with AKAP100 and immunolocalization of RI in cardiac myocytes. Freshly isolated adult rat left ventricular myocytes were double-stained with RII- ( A ) and AKAP100-specific antibodies ( B ), or with RI-specific antibody ( D ). The anti-AKAP100 rabbit antibodies were detected with LRSC-conjugated donkey anti–rabbit IgG antibodies. Localization of RI-specific monoclonal antibody and RII-specific goat antibodies were visualized by using FITC-conjugated donkey anti–mouse IgG antibodies and FITC-conjugated donkey anti–goat IgG antibodies, respectively. C is the superimposed image of A and B . The transverse periodic staining ( T ), sarcolemmal staining ( S ), and the nuclear staining ( N ) are indicated. In D , the longitudinal staining pattern is labeled with arrowheads, and the nucleus without staining is labeled with an N . Bar, 5 μm.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Isolation, Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Colocalization of RII with AKAP100 in cross-sections of cardiac myocytes. Transverse cryostat sections of rat papillary muscles were double-immunostained with RII-specific ( A ) and AKAP100-specific ( B ) antibodies. The primary antibodies were detected as described in Fig. . The arrows label the nuclear staining ( N ) for RII and for AKAP100. The arrowheads indicates the similar RII and AKAP100 network staining patterns. Bar, 5 μm.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Muscles, Staining

Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Localization of AKAP100 in the FITC-phalloidin–stained cardiac myocytes. Freshly isolated adult rat left ventricular myocytes were incubated with FITC-phalloidin ( A ) followed by immunostaining with AKAP100-specific rabbit antibodies ( B ). Localization of the anti-AKAP100 antibodies was detected with LRSC-conjugated donkey anti–rabbit IgG antibodies. C is the superimposed image of A and B . In A , the location of the Z-line at the center of phalloidin staining is indicated ( Z ), and the nucleus without staining is labeled with an N . In A and C , transverse periodic staining ( T ), sarcolemmal staining ( S ), and nuclear staining ( N ) are shown. Bar, 5 μm.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Staining, Isolation, Incubation, Immunostaining, Labeling

Journal: The Journal of Cell Biology

Article Title: A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

doi:

Figure Lengend Snippet: Colocalization of AKAP100 with α-actinin and RyR in cross-sections of cardiac myocytes. Cross-sections of the rat papillary muscle were double-immunostained with either α-actinin–specific ( A ) and AKAP100-specific ( B ) antibodies or RyR-specific ( D ) and AKAP100-specific ( E ) antibodies. The primary antibodies were detected as described in Fig. . C is the superimposed image of A and B ; F is combined from the images in D and E . The myocytes are labeled as in Fig. . In B , arrowheads indicate the network pattern of the AKAP100 staining, which is not seen in the α-actinin staining ( A ). In C , AKAP100 staining localized in the boundary of the α-actinin staining is indicated by arrowheads. In D and E , arrowheads indicate the similar RyR and AKAP100 network staining. In F , arrowheads indicate the punctate RyR staining that does not overlap with the AKAP100 staining. Bar, 5 μm.

Article Snippet: Polyclonal rabbit IgG antibodies against human AKAP100 recombinant protein and polyclonal goat IgG antibodies against mouse RII recombinant were obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Techniques: Labeling, Staining