The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 33 kDa (predicted molecular weight: 33 kDa).
Ser/Thr-kinase component of cyclin D-CDK4 (DC) complexes that phosphorylate and inhibit members of the retinoblastoma (RB) protein family including RB1 and regulate the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complexes and the subsequent transcription of E2F target genes which are responsible for the progression through the G(1) phase. Hypophosphorylates RB1 in early G(1) phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also phosphorylates SMAD3 in a cell-cycle-dependent manner and represses its transcriptional activity. Component of the ternary complex, cyclin D/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex.
Involvement in disease
Defects in CDK4 are a cause of susceptibility to cutaneous malignant melanoma type 3 (CMM3) [MIM:609048]. Malignant melanoma is a malignant neoplasm of melanocytes, arising de novo or from a pre-existing benign nevus, which occurs most often in the skin but also may involve other sites.
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily. Contains 1 protein kinase domain.
Phosphorylation at Thr-172 is required for enzymatic activity. Phosphorylated, in vitro, at this site by CCNH-CDK7, but, in vivo, appears to be phosphorylated by a proline-directed kinase. In the cyclin D-CDK4-CDKN1B complex, this phosphorylation and consequent CDK4 enzyme activity, is dependent on the tyrosine phosphorylation state of CDKN1B. Thus, in proliferating cells, CDK4 within the complex is phosphorylated on Thr-172 in the T-loop. In resting cells, phosphorylation on Thr-172 is prevented by the non-tyrosine-phosphorylated form of CDKN1B.
Cytoplasm. Nucleus. Membrane. Cytoplasmic when non-complexed. Forms a cyclin D-CDK4 complex in the cytoplasm as cells progress through G(1) phase. The complex accumulates on the nuclear membrane and enters the nucleus on transition from G(1) to S phase. Also present in nucleoli and heterochromatin lumps. Colocalizes with RB1 after release into the nucleus.
Lanes 1: Wild-type HAP1 cell lysate (20 µg) Lanes 2: CDK4 knockout HAP1 cell lysate (20 µg) Lanes 1 and 2: Merged (red and green) signal. Green signal from target – ab95255 observed at 34 kDa. Red signal from loading control – ab8226 observed at 42 kDa
ab95255 was shown to react with CDK4 when CDK4 knockout samples were used, along with additional cross-reactive bands. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab95255 and ab8226 (loading control to beta actin) were diluted 1 µg/mL and 1/2000 respectively and incubated overnight at 4ºC. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
Western blot - Anti-Cdk4 antibody (ab95255)
Anti-Cdk4 antibody (ab95255) at 1 µg/ml + MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg
Predicted band size: 33 kDa Observed band size: 33 kDa Additional bands at: 110 kDa, 16 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 20 minutes
A positive signal was also observed within HeLa whole cell lysate.
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