Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HIF1A KO HCT 116 (HIF1A knockout human colorectal carcinoma epithelial cell), ab255394 cells labelling HIF-1 alpha with AB308433 at 1/100 dilution (10.25 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green).
Confocal images showing nuclear staining in wildtype HCT 116 cells treated with DFO (0.5mM) for 24 hours and showing no staining in HIF1A knockout HCT 116 cells treated with the same condition. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml) (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 ug/ml).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling HIF-1 alpha with AB308433 at 1/100 dilution (5.125 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green).
Confocal images showing nuclear staining in HeLa cells treated with DFO (0.5 mM) for 24 hours and showing no staining in untreated HeLa cells.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml) (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 ug/ml).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized bEnd.3 (mouse brain endothelial cell) cells labelling HIF-1 alpha with AB308433 at 1/100 dilution (5.125 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green).
Confocal images showing nuclear staining in bEnd.3 cells treated with DFO (0.5 mM) for 24 hours and showing no staining in untreated bEnd.3 cells.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml) (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 ug/ml).

Blocking and diluting buffer and concentration: 5% NFDM/TBST.#Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

In Western blot, ab308433 was shown to bind specifically to HIF-1 alpha. A band was observed at 110 kDa in wild-type HCT116 cell lysates with whereas no signal observed at this size in HIF-1 alpha knockout cell line ab255394.

The expression of HIF-1 alpha is upregulated in response to DFO treatment (PMID: 25075254).

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

Exposure time: 180 seconds.

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

The expression of HIF-1 alpha is upregulated in response to DFO treatment (PMID: 25075254).

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.