All lanes : Anti-PAI1 antibody [EPR21850-82] (ab222754) at 1/1000 dilution

Lane 1 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 2 : Human liver lysate
Lane 3 : Mouse placenta lysate
Lane 4 : Rat placenta lysate
Lane 5 : Hepa1-6 (Mouse hepatoma epithelial cell line) whole cell lysate
Lane 6 : Human placenta lysate
Lane 7 : HUVEC (Human umbilical vein endothelial cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Lanes 6-7 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa

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.

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.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 cells serum-starved for 4 hours, treated with TGF-β (10 ng/ml) for 3 hours, and then with TGF-β (10 ng/ml) and BFA (300 ng/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-β 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.

All lanes : Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/1000 dilution

Lane 1 : Mouse placenta tissue lysate
Lane 2 : Mouse lung tissue lysate
Lane 3 : Mouse liver tissue lysate
Lane 4 : Rat placenta tissue lysate
Lane 5 : Rat lung tissue lysate
Lane 6 : 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
Additional bands at: 37 kDa (possible non-specific binding)


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.

All lanes : Anti-PAI1 antibody [EPR21850-82] (ab222754) at 1/1000 dilution

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast cell line) serum-starved for 4 hours, whole cell lysate
Lane 2 : NIH/3T3 serum-starved for 4 hours then treated with 10 ng/ml TGF ß1 (ab50036) for 3 hours, then with 10 ng/ml TGF ß1 (ab50036) and 300 ng/ml Brefeldin A (BFA) together for 18 hours, 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

Predicted band size: 45 kDa


Exposure time: 32 seconds

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.

All lanes : Anti-PAI1 antibody [EPR21850-82] (ab222754) at 1/1000 dilution

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast cell line) serum-starved for 18 hours, then collected the supernatant lysate
Lane 2 : NIH/3T3 serum-starved for 18 hours then treated with 10 ng/ml TGF ß1 (ab50036) for 24 hours, then collected the supernatant lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa


Exposure time: 26 seconds

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.