Recombinant Anti-eIF4E (phospho S209) antibody [EP2151Y] (ab76256)

Blocking buffer and concentration 2% BSA/TBST.
Diluting buffer and concentration 2% BSA/TBST.

Immunocytochemistry/Immunofluorescence analysis of untreated, 20% serum treated and 20% serum + LP treated NIH/3T3 cells labelling eIF4E (phospho S209) with ab76256 (left) and eIF4E with ab33766 (right) both at a dilution of 1/500.

Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

The image shows increased cytoplasmic staining after 20% serum treatment on NIH3T3 cells when compared with no serum treated cells. The LP treatment decreased the increased cytoplasmic staining caused by 20% serum.

ab33766 was used as a Pan control for ab76256. The results showed cytoplasmic staining on no serum, 20% serum and 20% serum +LP treated NIH3T3 cells.

ab76256 (purified) at 1/40 immunoprecipitating eIF4E (phospho S209) in HEK293 whole cell lysate. 10 ug of cell lysate was present in the input. For western blotting, a HRP-conjugated Veriblot for IP Detection Reagent (ab131366) (1/1,500) was used for detection. A rabbit monoclonal IgG (ab172730) was used intead of ab128913 as a negative control (Lane 3).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

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

Dot blot analysis of eIF4E (pS209) peptide (Lane 1) and eIF4E non-phospho peptide (Lane 2) labelling eIF4E (pS209) with purified ab76256 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

Immunocytochemistry/Immunofluorescence analysis of serum starved HEK293 cells treated with CGP 57380 ab120365) labelling eIF4E (phospho S209) with unpurified ab32124 at 1/100. Decrease in eIF4E (phospho S209) expression correlates with increased concentration of CGP 57380, as described in literature.
The cells were incubated at 37°C for 1h in media containing different concentrations of ab120365 (CGP 57380) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with unpurified ab76256 was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

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

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time:

eIF4E pS209: 15 seconds.

eIF4E: 3 minutes.

Blocking and dilution buffer: 5% NFDM/TBST.