MA5-14260

antibody from Invitrogen Antibodies

Targeting: CDKN2A ARF, CDK4I, CDKN2, CMM2, INK4, INK4a, MLM, MTS1, p14, p14ARF, p16, p16INK4a, p19, p19Arf

Provider product page for MA5-14260

Western blot

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [26]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [1]
Other assay [3]

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Product number: MA5-14260 - Provider product page
Provider: Invitrogen Antibodies
Product name: p14ARF Monoclonal Antibody (14P02 (DCS-240))
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA5-14260 targets p14ARF/p16beta in ICC/IF, IP, and WB applications and shows reactivity with Human samples.
Antibody clone number: 14P02 (DCS-240)
Concentration: 0.2 mg/mL

CDKN2A protein structure - MA5-14260 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of CDKN2A (p14ARF) was performed with 10 µg of HeLa cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against CDKN2A (p14ARF) (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. CDKN2A (p14ARF) was detected at ~ 14 kDa using CDKN2A (p14ARF) Mouse monoclonal antibody (Product # MA5-14260) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Mouse (Product # 31437) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to GAPDH (36 kDa). CDKN2A (p14ARF) Antibody (Product # MA5-14260) confirms silencing of CDKN2A (p14ARF) expression.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of p14ARF/p16beta using p14ARF/p16beta Monoclonal Antibody (Product # MA5-14260) on HeLa Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of CDKN2A was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042) (Assay ID CRISPR697306_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of CDKN2A was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2), HeLa CDKN2A KO (Lane 3) whole cell extracts. The blot was probed with Anti-p14ARF Monoclonal Antibody (14P02 (DCS-240))(Product # MA5-14260) using 2 µg/mL dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to CDKN2A. An uncharacterized band was observed at ~110 kDa and 70 kDa

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of p14ARF/p16beta using p14ARF/p16beta Monoclonal Antibody (Product # MA5-14260) on Native Human HeLa Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5. Androgen receptor interrupts ARF/MET axis. (A) AR inversely correlates with MET in prostate cancer cell lines. ARF levels were used as reference control based on our previous report ( 20 ). (B) ARF, MET and its signaling proteins beta-Catenin, SOX9 co-upregulation by androgen depletion in LAPC4 cells. LAPC4 cells were cultured in cFBS to deplete androgen for 6 days and regular FBS as control. (C) AR inversely correlates with MET in the prostate sphere. (D) ARF knockdown mediated MET downregulation is cytosolic MET-dependent. C, cytosolic lysates; N, nuclear lysates. (E) ARF knockdown mediated MET downregulation is androgen-dependent and is interrupted by AR activation. (F) AR overexpression and activation disrupts ARF and MET interactions. PC3 cells were transfected by AR and stimulated by DHT at 15nM for 24hrs. (G) ARF inducible expression in LNCaP cells increases androgen independent cell growth. GFP fusion ARF expression is detected by GFP expression by imaging (left panel) and cell growth were detected by crystal violet staining (right panel). (H) Nuclear MET stimulates LNCaP cells migration independent on androgen but mMET dependents on androgen. (I) AR correlates with DNA repair protein BRCA1 in nucleus. (J) ARF knockdown increases BRCA1 nuclear localization. (K) AR, nMET, and ARF regulate ATM and PARP but not caspase 3 investigated by western blot. Data are representative of averages + SD (standard deviation).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5. EBdCas9 upregulates CDKN2A ( p16 ) expression and compromises cell viability (A) Integrative genomic viewer of CDKN2A H3K27me3 and H3K4me3 promoter tiling. (B) Timeline of EBdCas9 or NCdCas9 induction and gRNA transfection. (C) qRT-PCR analysis of EBdCas9 or NCdCas9 after 3 days of transfection with single gRNA (g1-g8) normalized to beta-actin and relative fold change compared with no guide (-g). (D) TATA boxes/initiator representation with respect to surrounding guide loci using the Element Navigation Tool for detection of core promoter elements. (E) Immunofluorescent imaging of EBdCas9 after 3 days of transfection with single p16 gRNA 1 (+g1) or no transfection (-g). Blue, DAPI; green, p16; far red, p21; scale bar, 100 um. (F) Cell viability of EBdCas9 and NCdCas9 transfected with p16 g1 (+g1) compared with no guide (-g). Time points were analyzed every 6 h using Alamar Blue (n = 3, SEM; *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test). (G) EBdCas9 timeline for measuring epigenetic remodeling. (H) qRT-PCR of p16 relative fold change of EBdCas9 and NCdCas9 after 3 days of Dox induction and p16 g1 transfection. Samples were normalized to beta-actin and compared with no guide (-g) of each respective cell line. (I) ChIP-qPCR of induced (+Dox) EBdCas9 and NCdCas9 after 3 days of transfection with p16 g1 RNA (+g1) or no transfection (-g). Antibodies used for ChIP are listed above (Cas9, EZH2, H3K27me3). Normalized to fraction input/H3 (n = 3, SEM; *p < 0.05, **p