The second messenger cyclic AMP mediates diverse cellular responses to external signals such as proliferation, ion transport, regulation of metabolism and gene transcription by activation of the cAMP-dependent protein kinase (cAPK or PKA). Activation of PKA occurs when cAMP binds to the two regulatory subunits of the tetrameric PKA holoenzyme resulting in release of active catalytic subunits. One of several regulatory subunits, p-PKA IIβ reg (cAMP-dependent protein kinase type II-beta regulatory subunit), also known as PRKAR2B, is a 418 amino acid protein that is phosphorylated by the activated catalytic chain. p-PKA IIβ reg knockout mice exhibit diminished white adipose tissue and were protected against diet-induced obesity and fatty livers,as well as markedly reduced leptin mRNA. Also playing a role in the immune response, p-PKA IIβ reg suppresses CREB transcriptional activity and down-regulates IL-2 production in T-lymphocytes.
Background References
1. Corredor RG et al. Soluble adenylyl cyclase activity is necessary for retinal ganglion cell survival and axon growth. J Neurosci 32:7734-44 (2012).
2. Nagalla S et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 117:5189-97 (2011).
Sequence Similarity
Belongs to the cAMP-dependent kinase regulatory chain family.
Tissue Specificity
Four types of regulatory chains are found: I-alpha, I-beta, II-alpha, and II-beta. Their expression varies among tissues and is in some cases constitutive and in others inducible.
Western blot analysis of PRKAR2B (regulatory subunit) on rat brain tissue lysates with Rabbit anti-PRKAR2B (regulatory subunit) antibody (ET1704-15) at 1/2,000 dilution.
Lysates/proteins at 40 µg/Lane.
Predicted band size: 46 kDa Observed band size: 50 kDa
Exposure time: 1 minute; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1704-15) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-PRKAR2B (regulatory subunit) antibody (ET1704-15) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1704-15) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-PRKAR2B (regulatory subunit) antibody (ET1704-15) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1704-15) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ICC staining of PRKAR2B (regulatory subunit) in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1704-15, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ICC staining of PRKAR2B (regulatory subunit) in N2A cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1704-15, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ICC staining of PRKAR2B (regulatory subunit) in SH-SY5Y cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1704-15, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
Flow cytometric analysis of PRKAR2B (regulatory subunit) was done on N2A cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1704-15, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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