All lanes : Anti-FOXP4 antibody [EPR22714-2] (ab242127) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate
Lane 2 : FOXP4 knock out HepG2 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

Predicted band size: 73 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

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

The lysates were kindly provided by our collaborator Dr. Bin Zhao, Zhejiang University.

 

Lanes 1-2: Merged signal (red and green). Green - ab242127 observed at 80 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

 

Lanes 1-2: ab242127 Anti-FOXP4 antibody [EPR22714-2] was shown to react with FOXP4 in HepG2 cells in Western blot. Loss of signal was observed when FOXP4 knockout sample was used. Wild-type and FOXP4 knockout samples were subjected to SDS-PAGE. ab242127 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 dilution and 1 in 10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-FOXP4 antibody [EPR22714-2] (ab242127) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate
Lane 2 : Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : Hepa1-6 (mouse hepatoma epithelial cell) 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 (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 73 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

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

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time: Lanes 1-2: 59 seconds; Lanes 3-4: 3 minutes.

 

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling FOXP4 with ab242127 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human tonsil. The section was incubated with ab242127 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument, Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 cells labelling FOXP4 with ab242127 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HepG2 cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000  dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling FOXP4 with ab242127 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

FOXP4 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab242127 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab242127 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 ug

Lane 2: ab242127 IP in HepG2 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab242127 in HepG2 whole cell lysate

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

Exposure time: 8 seconds

 

All lanes : Anti-FOXP4 antibody [EPR22714-2] (ab242127) at 1/1000 dilution

Lane 1 : Human colon tissue lysate
Lane 2 : Human tonsil tissue lysate
Lane 3 : Rat spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-2 : VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/1000 dilution
Lane 3 : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 73 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

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

Bands around 50 kDa and 37 kDa in lane 3 are caused by degradation.

Exposure time: 3 minutes

 

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling FOXP4 with ab242127 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse spleen. The section was incubated with ab242127 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse colon tissue labeling FOXP4 with ab242127 at 1/1000 dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Nuclear staining on mouse colon is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

All lanes : Anti-FOXP4 antibody [EPR22714-2] (ab242127) at 1/1000 dilution

Lane 1 : 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma ) 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 (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 73 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

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

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time: 3 minutes

 

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling FOXP4 with ab242127 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat spleen. The section was incubated with ab242127 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.