All lanes : Anti-CDKN2A/p14ARF antibody [EPR17878] (ab185620) at 1/1000 dilution

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : CDKN2A knockout HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 14 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab185620 observed at 14 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab185620 was shown to specifically react with CDKN2A/p14ARF in wild-type HeLa cells as signal was lost in CDKN2A knockout cells. Wild-type and CDKN2A knockout samples were subjected to SDS-PAGE. Ab185620 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-CDKN2A/p14ARF antibody [EPR17878] (ab185620) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate transfected with empty vector (vector control)
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate transfected with CDKN2A/p14ARF with His-tag

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 14 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-CDKN2A/p14ARF antibody [EPR17878] (ab185620) at 1/1000 dilution

Lane 1 : PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 14 kDa
Observed band size: 14 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression level of p14 ARF is relatively low in most cell types and will be induced by oncogene expression. (PMID: 9694807; PMID: 9694806).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma) cells transfected with CDKN2A/p14ARF or empty vector, labeling CDKN2A/p14ARF with ab185620 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution 

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab185620 at 1/250 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells transfected with CDKN2A/p14ARF-GFP labeling CDKN2A/p14ARF with ab185620 at 1/120 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti Rabbit IgG (PE) at 1/150 dilution was used as the secondary antibody.

CDKN2A/p14ARF was immunoprecipitated from 1mg of CDKN2A/p14ARF transfected HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab185620 at 1/80 dilution.

Western blot was performed from the immunoprecipitate using ab185620 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: CDKN2A/p14ARF transfected HeLa whole cell lysate, 10µg (Input).

Lane 2: ab185620 IP in CDKN2A/p14ARF transfected HeLa whole cell lysate.

Lane 3: Rabbit IgG,monoclonal[EPR25A] - Isotype Control (ab172730) instead of ab185620 in CDKN2A/p14ARF transfected HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.