Anti-PAI1 antibody [EPR21850-82]

10 product images

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  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) (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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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

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

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

  Predicted band size: 45 kDa

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  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) (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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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.

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

  Predicted band size: 45 kDa

  Observed band size: 37 kDa, 45 kDa

  Exposure time: 3min

  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.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-HIF-1 alpha antibody [EPR16897] ab179483).

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  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 (Recombinant human TGF beta 1 protein (Active) ab50036) for 3 hours, then with 10 ng/ml TGF β1 (Recombinant human TGF beta 1 protein (Active) ab50036) 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 45 kDa

  Exposure time: 32s

• 

  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 (Recombinant human TGF beta 1 protein (Active) ab50036) for 24 hours, then collected the supernatant lysate at 10 µg

  Secondary

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

  Predicted band size: 45 kDa

  Exposure time: 26s

• 

  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) (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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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.

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  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (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).

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  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 Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed 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.

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  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.