All lanes : Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : SERPINE1 knockout A549 cell lysate
Lane 3 : HUVEC cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Lanes 1 - 3: Merged signal (red and green). Green - ab182973 observed at 48 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab182973 was shown to react with PAI1 in wild-type A549 cells in Western blot. The band observed in the edited lysate lane above 45 kDa is likely to represent SERPINE1 with an insertion. This has not been investigated further. Wild-type A549 and SERPINE1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab182973 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Hepa1-6 (mouse hepatoma epithelial cell line) cells labeling PAI1 with ab182973 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Hepa1-6 cells.

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

-ve control: PBS only, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methonol-permeabilized Hepa1-6 (mouse hepatoma epithelial cell line) cells labeling PAI1 with ab182973 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

Lane 1 : Rat placenta tissue lysate
Lane 2 : Rat lung tissue lysate
Lane 3 : Rat liver tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 20 seconds

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

The expression level of mouse and rat PAI1 may be low in normal liver tissue (PMID: 21898503). This antibody detects no bands in rat liver.

All lanes : Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

Lane 1 : Mouse placenta tissue lysate
Lane 2 : Mouse liver tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 45 kDa
Observed band size: 45 kDa

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

The expression level of mouse and rat PAI1 may be low in normal liver tissue (PMID: 21898503). This antibody detects a 37kDa extra band without BOI in mouse liver.

Exposure time:

Lane 1 : 3.25 seconds
Lane 2 : 180 seconds

All lanes : Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

Lane 1 : Hepa1-6 (mouse hepatoma epithelial cell line) whole cell lysate
Lane 2 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 45 kDa
Observed band size: 45 kDa

Blocking and dilution buffer: 5% NFDM/TBST

Exposure times.

Lane 1: 1 second

Lane 2: 3 seconds

Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution + Mouse lung (7 days supernatant) at 10 µl

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

Developed using the ECL technique.

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 3 seconds

Blocking and dilution buffer: 5% NFDM/TBST 

All lanes : Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/5000 dilution

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) grown in serum-free media for 18 hours, whole cell lysate
Lane 2 : HepG2 grown in serum-free media for 18 hours, then treated with 10ng/ml TGFß for 24 hours, 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

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 180 seconds

Blocking and diluting buffer: 5% NFDM/TBST 

This image was performed on a BIO-RAD^® ChemiDoc™ MP instrument. The expression profile observed is consistent with what has been described in the literature (PMID: 20519507).