Lysine-specific demethylase 5A is an enzyme that in humans is encoded by the KDM5A gene. The protein encoded by this gene is a ubiquitously expressed nuclear protein. It binds directly, with several other proteins, to retinoblastoma protein which regulates cell proliferation. It was formerly known as Retinoblastoma Binding Protein 2 (RBP2). This protein also interacts with rhombotin-2 which functions distinctly in erythropoiesis and in T-cell leukemogenesis. Rhombotin-2 is thought to either directly affect the activity of the encoded protein or may indirectly modulate the functions of the retinoblastoma protein by binding to this protein. Alternatively spliced transcript variants encoding distinct isoforms have been found for this gene. The Drosophila homolog, LID, was found to be an H3K4 histone demethylase that binds to c-Myc. It was recently renamed to Lysine Demethylase 5 (KDM5). Enzymatically can be designated as a trimethyllysine dioxygenase, which is a member of the alpha-ketoglutarate-dependent hydroxylase superfamily (EC 1.14.11.8).
Background References
1. Chan S W et al. Retinoblastoma-binding protein 2 (Rbp2) potentiates nuclear hormone receptor-mediated transcription. J Biol Chem 276:28402-28412 (2001).
2. Benevolenskaya E V et al. Binding of pRB to the PHD protein RBP2 promotes cellular differentiation. Mol Cell 18:623-635 (2005).
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7107-43) at 1/2,000 dilution was used in K1803 at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
ICC staining of KDM5A in A549 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 (ET7107-43, 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 KDM5A in SiHa 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 (ET7107-43, 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 KDM5A was done on SH-SY5Y cells. The cells were fixed, permeabilized and stained with the primary antibody (ET7107-43, 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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