Anti-PKN1 antibody [EPR18808]

• IP

Supplier Data

Immunoprecipitation - Anti-PKN1 antibody [EPR18808] (AB195264)

PKN1 was immunoprecipitated from 1mg of Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate with ab195264 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab195264 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Jurkat whole cell lysate, 10μg (Input).

Lane 2 : ab195264 IP in Jurkat whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal-Isotype Control (ab172730) instead of ab195264 in Jurkat whole cell lysate.

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-PKN1 antibody [EPR18808] (ab195264)

Predicted band size: 104 kDa

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• WB

Lab

Western blot - Anti-PKN1 antibody [EPR18808] (AB195264)

Lane 1 : Wild-type HAP1 cell lysate (40 μg)
Lane 2 : PKN1 knockout HAP1 cell lysate (40 μg)
Lane 3 : MCF7 cell lysate (20 μg)
Lane 4 : K562 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab195264 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab195264 was shown to specifically react with PKN1 when PKN1 knockout samples were used. Wild-type and PKN1 knockout samples were subjected to SDS-PAGE. ab195264 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PKN1 antibody [EPR18808] (ab195264)

Predicted band size: 104 kDa

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• WB

Supplier Data

Western blot - Anti-PKN1 antibody [EPR18808] (AB195264)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PKN1 antibody [EPR18808] (ab195264) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 2:

LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg

Lane 3:

MDA-MB-231 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 4:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 20 µg

Lane 5:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 6:

MCF-7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 104 kDa

Observed band size: 120 kDa

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Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-PKN1 antibody [EPR18808] (AB195264)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PKN1 antibody [EPR18808] (ab195264) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 104 kDa

Observed band size: 120 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-PKN1 antibody [EPR18808] (AB195264)

All lanes:

Western blot - Anti-PKN1 antibody [EPR18808] (ab195264) at 1/1000 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

Rat spleen lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 104 kDa

Observed band size: 120 kDa

false

Exposure time: 3min

• WB

CiteAb

Western blot - Anti-PKN1 antibody [EPR18808] (AB195264)

Western Blotting using Anti-PKN1 antibody [EPR18808], ab195264. Publication image from Chang, J. et al., 2018, Nat Commun, 30206226. Legend direct from paper.

PKN1 rs34309238 variant influences pancreatic cancer risk by altering the level of phosphorylated PKN1 and thus affecting the FAK/PI3K/AKT signalling pathway. a Protein modification sites of PKN1. Annotations were obtained from the PhosphoSitePlus database. b Result of the iTRAQ-based comparative proteomics screen. PANC-1 cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector. The raw intensity values of cells transfection with PKN1[A] or PKN1[C] were divided by the intensity values of cells transfection with control vector to obtain the relative intensity values. The y axis shows the relative intensity values of cells' transfection with PKN1[A] minus the relative intensity values of cells' transfection with PKN1[C]. The x axis shows the molecular weight of detected peptides. The proteomics screen experiment was repeated independently for two times with similar results. c Levels of phosphorylated FAK and AKT were affected by the PKN1 rs34309238 variant. Cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector (left) and PKN1-targeting siRNAs or control siRNA (right). d Levels of phosphorylated FAK and AKT were reduced by the PKN1 inhibitors. Cells were seeded in six-well plates after transfection with PKN1 inhibitors Lestaurtinib and Ro318220 or DMSO as control. For c, d, the western blot experiment was repeated independently for three times with similar results

false

• WB

CiteAb

Western blot - Anti-PKN1 antibody [EPR18808] (AB195264)

Western Blotting using Anti-PKN1 antibody [EPR18808], ab195264. Publication image from Chang, J. et al., 2018, Nat Commun, 30206226. Legend direct from paper.

PKN1 rs34309238 variant influences pancreatic cancer risk by altering the level of phosphorylated PKN1 and thus affecting the FAK/PI3K/AKT signalling pathway. a Protein modification sites of PKN1. Annotations were obtained from the PhosphoSitePlus database. b Result of the iTRAQ-based comparative proteomics screen. PANC-1 cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector. The raw intensity values of cells transfection with PKN1[A] or PKN1[C] were divided by the intensity values of cells transfection with control vector to obtain the relative intensity values. The y axis shows the relative intensity values of cells' transfection with PKN1[A] minus the relative intensity values of cells' transfection with PKN1[C]. The x axis shows the molecular weight of detected peptides. The proteomics screen experiment was repeated independently for two times with similar results. c Levels of phosphorylated FAK and AKT were affected by the PKN1 rs34309238 variant. Cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector (left) and PKN1-targeting siRNAs or control siRNA (right). d Levels of phosphorylated FAK and AKT were reduced by the PKN1 inhibitors. Cells were seeded in six-well plates after transfection with PKN1 inhibitors Lestaurtinib and Ro318220 or DMSO as control. For c, d, the western blot experiment was repeated independently for three times with similar results

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