All lanes : Anti-PHD3 antibody [EPR17869] (ab184714) at 1/10000 dilution

Lane 1 : PHD3 transfected HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysate
Lane 2 : Empty vector (vector control) transfected HEK-293 whole cell lysate
Lane 3 : PHD3 transfected HEK-293 whole cell lysate treated with 0.1 mM CoCl2 for 4 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 27 kDa
Observed band size: 27 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-PHD3 antibody [EPR17869] (ab184714) at 1/2000 dilution + Human fetal liver lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 27 kDa
Observed band size: 27 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-PHD3 antibody [EPR17869] (ab184714) at 1/5000 dilution + A549 (Human lung carcinoma) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 27 kDa
Observed band size: 27 kDa


Exposure time: 2 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PHD3 antibody [EPR17869] (ab184714) at 1/2000 dilution

Lane 1 : Mouse pancreas lysate
Lane 2 : Rat pancreas lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 27 kDa
Observed band size: 27 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PHD3 antibody [EPR17869] (ab184714) at 1/2000 dilution

Lane 1 : Mouse kidney lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Rat brain lysate
Lane 4 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 6 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 27 kDa
Observed band size: 27 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PHD3 antibody [EPR17869] (ab184714) at 1/2000 dilution

Lane 1 : Untreated MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : MCF7 cell lysate treated with 0.5mM CoCl2 (Cobalt (II) chloride) for 6 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 27 kDa
Observed band size: 27 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

PHD3 expression was induced by CoCl2 treatment (PMID: 18337469).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (Human lung carcinoma) cells labeling PHD3 with ab184714 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing weakly cytoplasm and nuclear staining on A549 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab184714 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-12 (Rat adrenal gland pheochromocytoma) cells labeling PHD3 with ab184714 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing weakly cytoplasm and nuclear staining on PC-12 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab184714 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

PHD3 was immunoprecipitated from 1mg of NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate with ab184714 at 1/70 dilution.

Western blot was performed from the immunoprecipitate using ab184714 at 1/1000 dilution.

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

Lane 1: NIH/3T3 whole cell lysate 10ug (Input).

Lane 2: ab184714 IP in NIH/3T3 whole cell lysate.

Lane 3: NIH/3T3 whole cell lysate supernatant after capture (unbound). 

Lane 4: Rabbit monoclonal IgG (ab172730) instead of ab184714 in NIH/3T3 whole cell lysate.

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

Exposure time: 30 seconds.

 

ab184714 is not a strong binder for IP - only a partial amount of the target protein in the lysate was immune-precipitated.