Anti-PAI1 antibody [EPR17272-21]

• IP

Lab

Immunoprecipitation - Anti-PAI1 antibody [EPR17272-21] (AB182973)

ab182973 at 1/30 immunoprecipitating PAI1 in SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate. Lane 1 (input) : SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate (10µg) Lane 2 (+) : SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab182973 in SW480 whole cell lysate For western blotting, ab182973 followed by ab131366 VeriBlot for IP (HRP) was used for detection (1/5000). Blocking/Diluting buffer and concentration : 5% NFDM/TBST.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PAI1 antibody [EPR17272-21] (AB182973)

Intracellular 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR17272-21] (AB182973)

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.

• IP

Lab

Immunoprecipitation - Anti-PAI1 antibody [EPR17272-21] (AB182973)

ab182973 at 1/30 immunoprecipitating PAI1 in SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate. Lane 1 (input) : Mouse placenta tissue lysate 10 μg Lane 2 (+) : Mouse placenta tissue lysate Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab182973 in mouse placenta tissue lysate For western blotting, ab182973 followed by ab131366 VeriBlot for IP (HRP) were used for detection (1/5000). Blocking/Diluting buffer and concentration : 5% NFDM/TBST.

Lanes 1 - 3:

Immunoprecipitation - Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/30 dilution

Lanes 1 - 3:

Immunoprecipitation - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/pai1-antibody-epr17272-21-bsa-and-azide-free-ab226864'>ab226864</a>)

All lanes:

Mouse placenta tissue lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 8s

• WB

Supplier Data

Western blot - Anti-PAI1 antibody [EPR17272-21] (AB182973)

Blocking and dilution buffer : 5% NFDM/TBST

Exposure times.

Lane 1 : 1 second

Lane 2 : 3 seconds

All lanes:

Western blot - Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

Lane 1:

Hepa1-6 (mouse hepatoma epithelial cell line) whole cell lysate at 10 µg

Lane 2:

HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

true

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR17272-21] (AB182973)

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:

Western blot - Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

Lane 1:

Rat placenta tissue lysate at 20 µg

Lane 2:

Rat lung tissue lysate at 20 µg

Lane 3:

Rat liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR17272-21] (AB182973)

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.

All lanes:

Western blot - Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

SERPINE1 knockout A549 cell lysate at 20 µg

Lane 3:

HUVEC cell lysate at 20 µg

Predicted band size: 45 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR17272-21] (AB182973)

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).

All lanes:

Western blot - 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 at 20 µg

Lane 2:

HepG2 grown in serum-free media for 18 hours, then treated with 10ng/ml TGFβ for 24 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-PAI1 antibody [EPR17272-21] (AB182973)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

All lanes:

Mouse lung (7 days supernatant) at 10 µL

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

true

Exposure time: 3s

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR17272-21] (AB182973)

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:

Western blot - Anti-PAI1 antibody [EPR17272-21] (ab182973) at 1/1000 dilution

Lane 1:

Mouse placenta tissue lysate at 20 µg

Lane 2:

Mouse liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false