Anti-PAI1 antibody [EPR21850-82]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR21850-82] (AB222754)

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 Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PAI1 antibody [EPR21850-82] (AB222754)

Flow cytometry overlay histogram showing left HUVEC positive cells and right negative HEK293 stained with ab222754 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab222754) (1x 106 in 100μl at 0.2μg/ml (1/9900)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in HUVEC Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR21850-82] (AB222754)

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.

• IP

Unknown

Immunoprecipitation - Anti-PAI1 antibody [EPR21850-82] (AB222754)

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:

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

Predicted band size: 45 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR21850-82] (AB222754)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PAI1 antibody [EPR21850-82] (AB222754)

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).

• WB

Supplier Data

Western blot - 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.

All lanes:

Western blot - Anti-PAI1 antibody [EPR21850-82] (ab222754) at 1/1000 dilution

Lane 1:

HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Human liver lysate at 20 µg

Lane 3:

Mouse placenta lysate at 20 µg

Lane 4:

Rat placenta lysate at 20 µg

Lane 5:

Hepa1-6 (Mouse hepatoma epithelial cell line) whole cell lysate at 20 µg

Lane 6:

Human placenta lysate at 20 µg

Lane 7:

HUVEC (Human umbilical vein endothelial cell line) whole cell lysate at 20 µg

Secondary

Lanes 1 - 5:

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

Lanes 6 - 7:

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

false

• WB

Supplier Data

Western blot - Anti-PAI1 antibody [EPR21850-82] (AB222754)

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:

Western blot - 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 at 10 µg

Lane 2:

NIH/3T3 serum-starved for 4 hours then treated with 10 ng/ml TGF β1 (<a href='/en-us/products/proteins-peptides/recombinant-human-tgf-beta-1-protein-active-ab50036'>ab50036</a>) for 3 hours, then with 10 ng/ml TGF β1 (<a href='/en-us/products/proteins-peptides/recombinant-human-tgf-beta-1-protein-active-ab50036'>ab50036</a>) and 300 ng/ml Brefeldin A (BFA) together for 18 hours, 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

false

Exposure time: 32s

• WB

Supplier Data

Western blot - Anti-PAI1 antibody [EPR21850-82] (AB222754)

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.

All lanes:

Western blot - 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 at 10 µg

Lane 2:

NIH/3T3 serum-starved for 18 hours then treated with 10 ng/ml TGF β1 (<a href='/en-us/products/proteins-peptides/recombinant-human-tgf-beta-1-protein-active-ab50036'>ab50036</a>) for 24 hours, then collected the supernatant 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 1/100000 dilution

Predicted band size: 45 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR21850-82] (AB222754)

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:

Western blot - Anti-PAI1 antibody [EPR21850-82] (ab222754) at 1/1000 dilution

Lane 1:

Mouse placenta tissue lysate at 20 µg

Lane 2:

Mouse lung tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Rat placenta tissue lysate at 20 µg

Lane 5:

Rat lung tissue lysate at 20 µg

Lane 6:

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: 37 kDa,45 kDa

false

Exposure time: 3min