Recombinant Anti-PAI1 antibody [EPR21850-82] (ab222754)

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1-4 and 6: 3 minutes; Lane 5: 37 seconds: Lane 7: 8 seconds.

PAI1 forms complex with its target protease, t-PA (lane 7). The molecular mass observed is consistent with what has been described in the literature (PMID 21596853).

Lanes 6 and 7 were developed with a high sensitivity ECL substrate.

ab222754 staining SERPINE1 in HUV-EC cells, with negative expression in HEK293 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab222754 at 0.1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HUVEC (Human umbilical vein endothelial cell line) cells labeling PAI1 with ab222754 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1,000 dilution (green). Confocal image showing cytoplasmic staining in HUVEC cell line. The nuclear counter stain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 cells serum-starved for 4 hours, treated with TGF-ß (10ng/ml) for 3 hours, and then with TGF-ß (10ng/ml) and BFA (300ng/ml) together for 18 hours (Red) / Untreated control (Green) labeling PAI1 with ab222754 at 1/60 (red/green) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody. 

The expression of PAI-1 is induced by TGF-ß in NIH/3T3 cell line (PMID 17890327). 

PAI1 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell line) whole cell lysate with ab222754 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab222754 at 1/1,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution.
Lane 1: HepG2 whole cell lysate 10 µg (Input).
Lane 2: ab222754 IP in HepG2 whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab222754 in HepG2 whole cell lysate (-).
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.

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

The expression levels of mouse and rat PAI1 may be low in normal liver tissue (PMID: 21898503). This antibody detects a 37 kDa extra band and no specific band in mouse liver and no bands in rat liver.
Although lung tissue is reported to be PAI1 positive (PMID: 21768189, PMID: 17032919), this antibody can’t detect band of target in mouse lung and detects weak target band in rat lung.

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression of PAI-1 is induced by TGF-β in the NIH/3T3 cell line (PMID 17890327). The 110 kDa band likely represents PAI-1 in complex with its target protease, t-PA (PMID 21596853).

The blot was developed with a high sensitivity ECL substrate.

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression of PAI-1 is induced by TGF-β in the NIH/3T3 cell line (PMID 17890327).

The blot was developed with a high sensitivity ECL substrate.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PAI1 with ab222754 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1,000 dilution (green). Confocal image showing the signal is increased in 4 hour serum-starved NIH/3T3 cells treated with TGF-β (10 ng/ml) for 3 hours, then with TGF-β (10 ng/ml) and BFA (300 ng/ml) together for 18 hours. The expression of PAI-1 is induced by TGF-β in the NIH/3T3 cell line (PMID 17890327). The nuclear counter stain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.