All lanes : Anti-CEBP Beta antibody [E299] - C-terminal (ab32358) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CEBP Beta knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 36 kDa

Lanes 1-3: Merged signal (red and green). Green - ab32358 observed at 40 and 45 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab32358 Anti-CEBP Beta antibody [E299] - C-terminal was shown to specifically react with CEBP Beta in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261771 (knockout cell lysate ab256874) was used. Wild-type and CEBP Beta knockout samples were subjected to SDS-PAGE. ab32358 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 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 20000 dilution for 1 hour at room temperature before imaging.

 

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling CEBP Beta with Purified ab32358 at 1:500 dilution (1.2 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

All lanes : Anti-CEBP Beta antibody [E299] - C-terminal (ab32358) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CEBPB knockout HAP1 whole cell lysate
Lane 3 : U-87 MG whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 36 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab32358 observed at 45, 42, 20 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab32358 was shown to recognize CEBPB in wild-type HAP1 cells as signal was lost at the expected MW in CEBPB knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CEBPB knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab32358 and ab130007 (Mouse anti-Vinculin 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.

Band of interest at 45, 42, 20 corresponding to C/EBPbeta-FL, C/EBPbeta-LAP and C/EBPbeta-LIP respectively.

Anti-CEBP Beta antibody [E299] - C-terminal (ab32358) at 1/1000 dilution + NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates 15 at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 36 kDa

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling CEBP Beta with Purified ab32358 at 1:600 dilution (1 µg/ml) (red). Cells were fixed with 80% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

3T3-L1 (Mouse embryonic fibroblast cell line) cells were plated on cover slips, grown to post confluency and treated with adipogenic cocktail for 16 h. Cells were washed briefly with phosphate buffered saline (PBS) and fixed in methanol at −20°C for 5 min. Cells were then blocked with 1% BSA for 30 min before incubation with ab32358 at a 1/100 dilution at 4°C overnight and incubated with Alexa Fluor^® 488 goat anti-rabbit secondary antibodies (1∶500) for 1 h at room temperature.

DAPI staining was used for visualizing the nuclei.

Images were acquired with an Olympus FlowView FV1000.

ab32358 (purified) at 1:30 dilution (2µg) immunoprecipitating CEBP Beta in NIH/3T3 whole cell lysate.
Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10µg
Lane 2 (+): ab32358 & NIH/3T3 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32358 in NIH/3T3 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Anti-CEBP Beta antibody [E299] - C-terminal (ab32358) + MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 36 kDa

Anti-CEBP Beta antibody [E299] - C-terminal (ab32358) at 1/1000 dilution + PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates 15ug at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 36 kDa

Anti-CEBP Beta antibody [E299] - C-terminal (ab32358) at 1/1000 dilution + PC-12 (Rat adrenal gland pheochromocytoma cell line) cell lysate.

Predicted band size: 36 kDa



Observed bands
LAP*: 38kDa
LAP: 35kDa
LIP: 20kDa

All lanes : Anti-CEBP Beta antibody [E299] - C-terminal (ab32358) at 1/500 dilution

Lane 1 : NIH/3T3 (Mouse embryo fibroblast cell line) cells
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) cells
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) cells
Lane 4 : Rat Spleen Lysate
Lane 5 : Rat Kidney Lysate
Lane 6 : Rat Heart Lysate
Lane 7 : Rat Brain Lysate
Lane 8 : Mouse Spleen Lysate
Lane 9 : Mouse Kidney Lysate
Lane 10 : Mouse Heart Lysate
Lane 11 : Mouse Brain Lysate

Predicted band size: 36 kDa

ab32358, at a 1/50 dilution, staining CEBP Beta in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by Immunofluorescence.

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with ab32358 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32358, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.