CDK4 Antibody - #DF6102

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製品: CDK4 Antibody
カタログ: DF6102
タンパク質の説明: Rabbit polyclonal antibody to CDK4
アプリケーション: WB IHC
反応性: Human, Mouse, Rat
予測: Pig, Zebrafish, Bovine, Horse, Sheep, Rabbit, Dog, Xenopus
分子量: 34kDa; 34kD(Calculated).
ユニプロット: P11802
RRID: AB_2838070

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説明書
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WB Handbook
IHC Protocol
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製品説明

ソース:
Rabbit
アプリケーション:
WB 1:500-1:2000, IHC 1:50-1:200
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

反応性:
Human,Mouse,Rat
予測:
Pig(100%), Zebrafish(100%), Bovine(100%), Horse(100%), Sheep(100%), Rabbit(100%), Dog(100%), Xenopus(100%)
クローナリティ:
Polyclonal
特異性:
CDK4 Antibody detects endogenous levels of total CDK4.
RRID:
AB_2838070
引用形式: Affinity Biosciences Cat# DF6102, RRID:AB_2838070.
コンジュゲート:
Unconjugated.
精製:
The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).
保存:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
別名:

折りたたみ／展開

Cdk 4; cdk4; CDK4 protein; CDK4_HUMAN; Cell division kinase 4; Cell division protein kinase 4; CMM 3; CMM3; Crk3; Cyclin dependent kinase 4; Cyclin-dependent kinase 4; Melanoma cutaneous malignant 3; MGC14458; p34 cdk4; PSK J3; PSK-J3;

免疫原

免疫原:
Uniprot:

P11802(CDK4_HUMAN) >>Visit HPA database.

遺伝子(ID):
CDK4(1019)
タンパク質の説明:
Cyclin-dependent kinase activity is regulated by T-loop phosphorylation (Thr172 in the case of CDK4), by the abundance of their cyclin partners, and by association with CDK inhibitors of the Cip/Kip or INK family of proteins (1). The inactive ternary complex of CDK4/cyclin D and p27 Kip1/Cip1 requires extracellular mitogenic stimuli for the release and degradation of p27, which affects progression through the restriction point and pRb-dependent entry into S-phase (2). The active complex of CDK4/cyclin D targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (3). In HeLa cells, upon UV irradiation, upregulation of p16 INK4A association with CDK4/cyclin D3 leads to a G2 delay, implicating CDK4/cyclin D3 activity in progression through the G2-phase of the cell cycle (4).
タンパク質配列:
MATSRYEPVAEIGVGAYGTVYKARDPHSGHFVALKSVRVPNGGGGGGGLPISTVREVALLRRLEAFEHPNVVRLMDVCATSRTDREIKVTLVFEHVDQDLRTYLDKAPPPGLPAETIKDLMRQFLRGLDFLHANCIVHRDLKPENILVTSGGTVKLADFGLARIYSYQMALTPVVVTLWYRAPEVLLQSTYATPVDMWSVGCIFAEMFRRKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPPRGPRPVQSVVPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPE

種類予測

種類予測:

Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Species
Results
Score
Pig
100
Horse
100
Bovine
100
Sheep
100
Dog
100
Xenopus
100
Zebrafish
100
Rabbit
100
Chicken
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - P11802 基板として

Site PTM Type Enzyme
A2 Acetylation
Y17 Phosphorylation P07948 (LYN) , P07947 (YES1) , P12931 (SRC)
K22 Ubiquitination
K35 Ubiquitination
R101 Methylation
K106 Ubiquitination
K118 Ubiquitination
K142 Ubiquitination
K155 Ubiquitination
T172 Phosphorylation P50613 (CDK7)
T177 Phosphorylation
K211 Ubiquitination
T277 Phosphorylation
K282 Ubiquitination
K297 Ubiquitination

PTMs - P11802 酵素として

Substrate Site Source
P04179 (SOD2) S106 Uniprot
P06400 (RB1) T5 Uniprot
P06400 (RB1) S249 Uniprot
P06400 (RB1) T252 Uniprot
P06400 (RB1) T356 Uniprot
P06400 (RB1) T373 Uniprot
P06400 (RB1) S608 Uniprot
P06400 (RB1) S612 Uniprot
P06400 (RB1) S780 Uniprot
P06400 (RB1) S788 Uniprot
P06400 (RB1) S795 Uniprot
P06400 (RB1) S807 Uniprot
P06400 (RB1) S811 Uniprot
P06400 (RB1) T826 Uniprot
P06748 (NPM1) T199 Uniprot
P15923 (TCF3) S139 Uniprot
P15923 (TCF3) S245 Uniprot
P17480 (UBTF) S484 Uniprot
P28749 (RBL1) T369 Uniprot
P28749 (RBL1) S640 Uniprot
P28749 (RBL1) S650 Uniprot
P28749-1 (RBL1) S964 Uniprot
P28749 (RBL1) S975 Uniprot
P38398 (BRCA1) S632 Uniprot
P43694 (GATA4) S105 Uniprot
P84022-1 (SMAD3) T8 Uniprot
P84022 (SMAD3) T179 Uniprot
P84022 (SMAD3) S204 Uniprot
P84022 (SMAD3) S208 Uniprot
P84022-1 (SMAD3) S213 Uniprot
Q06830 (PRDX1) T90 Uniprot
Q08050 (FOXM1) S4 Uniprot
Q08050 (FOXM1) S35 Uniprot
Q08050 (FOXM1) S451 Uniprot
Q08050 (FOXM1) S489 Uniprot
Q08050 (FOXM1) S508 Uniprot
Q08050 (FOXM1) T510 Uniprot
Q08050 (FOXM1) S522 Uniprot
Q08050 (FOXM1) T600 Uniprot
Q08050 (FOXM1) T611 Uniprot
Q08050 (FOXM1) T620 Uniprot
Q08050 (FOXM1) T627 Uniprot
Q08050 (FOXM1) S704 Uniprot
Q08999 (RBL2) T401 Uniprot
Q08999 (RBL2) S672 Uniprot
Q08999 (RBL2) S1035 Uniprot
Q12778 (FOXO1) S249 Uniprot
Q13761 (RUNX3) S356 Uniprot
Q14814 (MEF2D) S98 Uniprot
Q14814 (MEF2D) S110 Uniprot
Q8IZL8 (PELP1) S477 Uniprot
Q8IZL8 (PELP1) S991 Uniprot
Q92879 (CELF1) S302 Uniprot
Q96KS0 (EGLN2) S130 Uniprot
Q9BQA1 (WDR77) T5 Uniprot
Q9BQA1 (WDR77) S264 Uniprot
Q9BQA1 (WDR77) S306 Uniprot
Q9NS23-2 (RASSF1) S203 Uniprot
Q9NS23 (RASSF1) S207 Uniprot

研究背景

機能:

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.

PTMs:

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.

細胞の位置付け:

Cytoplasm. Nucleus. Nucleus membrane.
Note: 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.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
サブユニット構造:

Component of the D-CDK4 complex, composed of CDK4 and some D-type G1 cyclin (CCND1, CCND2 or CCND3). Interacts directly in the complex with CCND1, CCND2 or CCND3. Interacts with SEI1 and ZNF655. Forms a ternary complex, cyclin D-CDK4-CDKN1B, involved in modulating CDK4 enzymatic activity. Interacts directly with CDKN1B (phosphorylated on 'Tyr-88' and 'Tyr-89'); the interaction allows assembly of the cyclin D-CDK4 complex, Thr-172 phosphorylation, nuclear translocation and enhances the cyclin D-CDK4 complex activity. CDK4 activity is either inhibited or enhanced depending on stoichiometry of complex. The non-tyrosine-phosphorylated form of CDKN1B prevents T-loop phosphorylation of CDK4 producing inactive CDK4. Interacts (unphosphorylated form) with CDK2. Also forms ternary complexes with CDKN1A or CDKN2A. Interacts directly with CDKN1A (via its N-terminal); the interaction promotes the assembly of the cyclin D-CDK4 complex, its nuclear translocation and promotes the cyclin D-dependent enzyme activity of CDK4. Interacts with CCND1; the interaction is prevented with the binding of CCND1 to INSM1 during cell cycle progression. Probably forms a complex composed of chaperones HSP90 and HSP70, co-chaperones CDC37, PPP5C, TSC1 and client protein TSC2, CDK4, AKT, RAF1 and NR3C1; this complex does not contain co-chaperones STIP1/HOP and PTGES3/p23. Interacts with CEBPA (when phosphorylated). Interacts with FNIP1 and FNIP2.

タンパク質ファミリー:

Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily.

研究領域

· Cellular Processes > Cell growth and death > Cell cycle.   (View pathway)

· Cellular Processes > Cell growth and death > p53 signaling pathway.   (View pathway)

· Cellular Processes > Cell growth and death > Cellular senescence.   (View pathway)

· Cellular Processes > Cellular community - eukaryotes > Tight junction.   (View pathway)

· Environmental Information Processing > Signal transduction > PI3K-Akt signaling pathway.   (View pathway)

· Human Diseases > Drug resistance: Antineoplastic > Endocrine resistance.

· Human Diseases > Infectious diseases: Viral > Hepatitis B.

· Human Diseases > Infectious diseases: Viral > Measles.

· Human Diseases > Infectious diseases: Viral > Human papillomavirus infection.

· Human Diseases > Infectious diseases: Viral > HTLV-I infection.

· Human Diseases > Cancers: Overview > Pathways in cancer.   (View pathway)

· Human Diseases > Cancers: Overview > Viral carcinogenesis.

· Human Diseases > Cancers: Specific types > Pancreatic cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Glioma.   (View pathway)

· Human Diseases > Cancers: Specific types > Melanoma.   (View pathway)

· Human Diseases > Cancers: Specific types > Bladder cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Chronic myeloid leukemia.   (View pathway)

· Human Diseases > Cancers: Specific types > Small cell lung cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Non-small cell lung cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Breast cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Hepatocellular carcinoma.   (View pathway)

· Organismal Systems > Immune system > T cell receptor signaling pathway.   (View pathway)

参考文献

1). Blockage of EGFR/AKT and mevalonate pathways synergize the antitumor effect of temozolomide by reprogramming energy metabolism in glioblastoma. Cancer communications (London, England), 2023 (PubMed: 37920878) [IF=16.2]

Application: WB    Species: Human    Sample: TBD0220, U-87 MG, and U-87 MG-EGFR-vIII cells

FIGURE 3 EGFR/AKT pathway regulates mitochondrial respiration and proliferation in GBM cells. (A-B) TBD0220 (A), and U-87 MG-EGFR-vIII cells (B) were treated with DMSO or 5 μmol/L MK-2206 for 24 h. The mitochondrial functions were monitored by Seahorse XF Cell Mito Stress test. The OCR, basal respiration, proton leak, and ATP production rates were measured as illustrated. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (independent-sample Student's t-test for TBD0220; one-way ANOVA for U-87 MG). (C) ATP levels in TBD0220, U-87 MG, and U-87 MG-EGFR-vIII cells were analyzed after 24 h of treatments with DMSO or 5 μmol/L of MK-2206. ∗∗P < 0.01, ∗∗∗P < 0.001 (independent-sample Student's t-test for TBD0220; one-way ANOVA for U-87 MG). (D) The cell growth assay for TBD0220, U-87 MG, and U-87 MG-EGFR-vIII lines treated with DMSO, 5 μmol/L MK-2206, or 5 μmol/L MK-2206 plus 50 μmol/L ATP were performed. ∗∗∗P < 0.001 (two-way ANOVA). (E) The colony formation assay of TBD0220, U-87 MG, and U-87 MG-EGFR-vIII lines treated with DMSO or 1 μmol/L MK-2206. (F) Cell cycle distributions were analyzed by flow cytometry in TBD0220, U-87 MG, and U-87 MG-EGFR-vIII cells treated with DMSO or 5 μmol/L MK-2206. (G) Western blotting to show changes in expressions of CDK2, CDK4, CDK6, Cyclin D, RB, p-RB, and GAPDH in TBD0220, U-87 MG, and U-87 MG-EGFR-vIII cells treated with DMSO or MK-2206. Abbreviations: EGFR, epidermal growth factor receptor; AKT, AKT serine/threonine kinase 1; GBM, glioblastoma; ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; CDK2, cyclin-dependent kinase 2; CDK4, cyclin-dependent kinase 4; CDK6, cyclin-dependent kinase 6; OCR, oxygen consumption rate.
2). Precise editing of FGFR3-TACC3 fusion genes with CRISPR-Cas13a in glioblastoma. Molecular Therapy, 2021 (PubMed: 34274537) [IF=12.4]
3). TCA-phospholipid-glycolysis targeted triple therapy effectively suppresses ATP production and tumor growth in glioblastoma. Theranostics, 2023 (PubMed: 36276638) [IF=12.4]

Application: WB    Species: Human    Sample: TBD0220 and U87MG cells

Figure 3 Reduction in ATP production hinders cell proliferation and contributes to G1/S arrest in GBM. (A-B) The relative viability of TBD0220 (A) and U87MG (B) cells was measured using a CCK-8 kit (n = 3). (C-D) Colony formation assay to detect GBM cell growth (D), and the quantification of colony numbers (C) (n = 3). (E) Cell cycle analysis using flow cytometry after incubation with different treatments like EPIC (20 µM), AA (25 µM), or EPIC (20 µM) + AA (25 µM) for 48 h, and the results are plotted as a histogram (n = 3) (F) Representative western blotting showing the expression of p21 and Rb and their downstream targets. The results were normalized to Tubulin with the control group as 1. Protein expression was quantified by ImageJ. (G) Representative confocal images of CDK6 after the treatment with AA (25 µM), EPIC (20 µM), or EPIC (20 µM) + AA (25 µM) for 48 h (n = 6). (H) The quantitative analysis of fluorescence images of CDK6 (n = 6). All data are shown as the mean values ± SD, and p values are based on one-way or two-way ANOVA. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Scale bar = 20 μm.
4). Targeted p53 activation by saRNA suppresses human bladder cancer cells growth and metastasis. Journal of Experimental & Clinical Cancer Research, 2016 (PubMed: 27012825) [IF=11.3]

Application: WB    Species: human    Sample: T24 and EJ cells

Fig. 4 dsP53-285 inhibits Cyclin D1 and CDKs, and reversed EMT-associated genes expression. T24 and EJ cells were transfected with 50 nM of the indicated dsRNAs for 72 h. a Expression of Cyclin D1 and CDK4/6 mRNA was detected by real-time PCR. GAPDH served as a loading control. b Expression of Cyclin D1 and CDK4/6 protein was detected by Western blot. α-tubulin served as a loading control. c Expression of EMTassociated genes mRNA was detected by real-time PCR. GAPDH served as a loading control. d Expression of EMT-associated genes protein was detected by Western blot analysis. GAPDH served as a loading control. * P < 0.05, **P < 0.01 and ***P < 0.001 compared to dsControl group
5). Combination LSD1 and HOTAIR-EZH2 inhibition disrupts cell cycle processes and induces apoptosis in glioblastoma cells. PHARMACOLOGICAL RESEARCH, 2021 (PubMed: 34246782) [IF=9.3]
6). Histone acetylation plays an important role in MC-LR-induced apoptosis and cycle disorder in SD rat testicular cells. Chemosphere, 2020 (PubMed: 31683423) [IF=8.8]

Application: WB    Species: Rat    Sample: testicular tissue

Fig4. The effect of TSA and MC-LR on cell cycle-related genes and proteins 434 levels in co-cultured Sertoli-germ cells and SD rat testicular tissues. (A) 435 Expressions of cell cycle-related genes in vitro and (B) expressions of cell 436 cycle-related genes in vivo were detected by RT-qPCR. (C) Expressions of cell cycle-related proteins in vitro and (D) expressions of cell cycle-related proteins in 438 vivo were detected by western blotting. (E) Quantitative analysis of proteins 439 expression levels in vitro. (F) Quantitative analysis of proteins expression levels in 440 vivo. (*P<0.05 vs. the control group; #P<0.05 vs. the 36 µM MC-LR group or the 40 441 µg kg-1 MC-LR group)
7). Isoliquiritigenin suppresses the progression of malignant melanoma via targeting H2A.Z.1-E2F1 pathway. Biochemical pharmacology, 2023 (PubMed: 37863326) [IF=5.8]

Application: WB    Species: Human    Sample: A375 and SK-MEL-28 cell

Fig. 2. ISL inhibited cell cycle progression. Two melanoma cell lines (A375 and SK-MEL-28) were treated with different concentrations of ISL (0, 10, 20 μM) for 24 h. (A) Cell cycle distribution analysis by flow cytometry for 48 h. (B) The expression of Cyclin D1, CDK2, CDK4 and CHEK1 in A375 and SK-MEL-28 cell lines with ISL administration for 48 h. The data represented the mean ± SEM of three independent experiments,
8). Effects of metformin on Sonic hedgehog subgroup medulloblastoma progression: In vitro and in vivo studies. Frontiers in Pharmacology, 2022 (PubMed: 36278239) [IF=5.6]
9). miR-3619-5p inhibits prostate cancer cell growth by activating CDKN1A expression. ONCOLOGY REPORTS, 2017 (PubMed: 27878260) [IF=4.2]

Application: WB    Species: human    Sample:

(D) Expression of cyclin D1, CDK4 and CDK6 mRNA was determined by western blot analysis. α-Tublin served as a loading control. *
10). Overexpression of p53 activated by small activating RNA suppresses the growth of human prostate cancer cells. OncoTargets and Therapy, 2016 (PubMed: 26811691) [IF=4.0]

Application: WB    Species: human    Sample: human

Figure 2 dsP53-285 suppresses Cyclin D1–CDK4/6 expression in PCa cell lines. Notes: LNCaP and DU145 cells were transfected with the indicated dsRNAs at 50 nM for 72 hours or mock transfected for 72 hours. (A) Expression of Cyclin D1 and CDK4/6 in both PCa cell lines was assessed by real-time PCR. GAPDH served as a loading control. *P,0.05 and **P,0.01 compared with the mock group; #P,0.05 and ##P,0.01 compared with the dsControl group. (B) Expression of Cyclin D1 and CDK4/6 protein was determined by Western blotting. α-Tubulin served as a loading control. Abbreviations: CDK, cyclin-dependent kinase; PCa, prostate cancer; dsRNA, double-stranded RNA; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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