The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/50000. Detects a band of approximately 110 kDa (predicted molecular weight: 106 kDa).
1/250 - 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
1/50 - 1/100.
1/1000 - 1/100000.
应用说明Is unsuitable for Flow Cyt.
功能Key regulator of entry into cell division that acts as a tumor suppressor. Promotes G0-G1 transition when phosphorylated by CDK3/cyclin-C. Acts as a transcription repressor of E2F1 target genes. The underphosphorylated, active form of RB1 interacts with E2F1 and represses its transcription activity, leading to cell cycle arrest. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV39H1, KMT5B and KMT5C, leading to epigenetic transcriptional repression. Controls histone H4 'Lys-20' trimethylation. Inhibits the intrinsic kinase activity of TAF1. Mediates transcriptional repression by SMARCA4/BRG1 by recruiting a histone deacetylase (HDAC) complex to the c-FOS promoter. In resting neurons, transcription of the c-FOS promoter is inhibited by BRG1-dependent recruitment of a phospho-RB1-HDAC1 repressor complex. Upon calcium influx, RB1 is dephosphorylated by calcineurin, which leads to release of the repressor complex (By similarity). In case of viral infections, interactions with SV40 large T antigen, HPV E7 protein or adenovirus E1A protein induce the disassembly of RB1-E2F1 complex thereby disrupting RB1's activity.
组织特异性Expressed in the retina.
疾病相关Childhood cancer retinoblastoma Bladder cancer Osteogenic sarcoma
序列相似性Belongs to the retinoblastoma protein (RB) family.
结构域The Pocket domain binds to the threonine-phosphorylated domain C, thereby preventing interaction with heterodimeric E2F/DP transcription factor complexes.
翻译后修饰Phosphorylated by CDK6 and CDK4, and subsequently by CDK2 at Ser-567 in G1, thereby releasing E2F1 which is then able to activate cell growth. Dephosphorylated at the late M phase. SV40 large T antigen, HPV E7 and adenovirus E1A bind to the underphosphorylated, active form of pRb. Phosphorylation at Thr-821 and Thr-826 promotes interaction between the C-terminal domain C and the Pocket domain, and thereby inhibits interactions with heterodimeric E2F/DP transcription factor complexes. Dephosphorylated at Ser-795 by calcineruin upon calcium stimulation. CDK3/cyclin-C-mediated phosphorylation at Ser-807 and Ser-811 is required for G0-G1 transition. Phosphorylated by CDK1 and CDK2 upon TGFB1-mediated apoptosis. N-terminus is methylated by METTL11A/NTM1 (By similarity). Monomethylation at Lys-810 by SMYD2 enhances phosphorylation at Ser-807 and Ser-811, and promotes cell cycle progression. Monomethylation at Lys-860 by SMYD2 promotes interaction with L3MBTL1. Acetylation at Lys-873 and Lys-874 regulates subcellular localization, at least during keratinocytes differentiation.
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized MCF7(Human breast denocarcinoma cell line) cells, Serum starved and non-starved, labeling Rb (phospho T373) with Ab52975 at 1/100 dilution followed by Goat anti-Rabbit secondary IgG AlexaFluor®488 (ab150077) secondary antibody at 1/1000 dilution (green).
Confocal image showing nuclear staining on MCF7 cells.The number of positive cells increased after treatment with FBS (fetal bovine serum) for 48 hours, then decreased after Lambda Protein Phosphatase treatment (31℃ for 2hours ).
Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/10000 dilution
Lane 1 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) 2 day serum starved whole cell lysate. Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate. Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate. Membrane incubated with phosphatase.
Lysates/proteins at 15 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Dot Blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
Dot Blot analysis of Lane 1: Rb (pT373) phospho peptide and Lane 2: Rb non-phospho peptide labeling Rb (phospho T373) with ab52975 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 3 minutes.
Western blot - Rb (phospho T373) antibody [EP821Y] (ab52975)
All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/50000 dilution
Lane 1 : Jurkat cell lysate (untreated) Lane 2 : Jurkat cell lysate (treated with FBS)
Lysates/proteins at 10 µg per lane.
Secondary goat anti-rabbit HRP labelled at 1/2000 dilution