Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) 抗体 [EP656Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

Overlay histogram showing HeLa cells stained with unpurified ab40795 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab40795, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in human liver carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1 : PBS in place of primary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in HeLa (human cervix adenocarcinoma) cells, treated and untreated with Lambda Protein Phosphtase 31℃ for 5h by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody. ab7291 and ab150120 were used as counterstains for primary antibody ab75748 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1 : Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2 : Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

• IP

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Immunoprecipitation - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

ab40795 immunoprecipitating PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154). 10μg of HeLa (human cervix adenocarcinoma) whole cell lysate was incubated with primary antibody at a dilution of 1/40 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

Lane 1 : HeLa whole cell lysate (10ug)
Lane 2 : ab40795 IP in HeLa whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab40795 in HeLa (human cervix adenocarcinoma) whole cell lysate

All lanes:

Immunoprecipitation - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (ab40795)

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in mouse cerebral cortex tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1 : PBS in place of primary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in rat cerebral cortex tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1 : PBS in place of primary

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in the human cell line NIH/3T3 (mouse embryo) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/120. A goat anti rabbit IgG (Alexa Fluorr^® 488) at a dilution of 1/500 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• WB

Lab

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (ab40795) at 1/1000 dilution

Lane 1:

MCF7, grown in serum-free media overnight, whole cell lysate at 10 µg

Lane 2:

MCF7, grown in serum-free media overnight, then treated with EGF 1μg/ml for 10min, whole cell lysate at 10 µg

Lane 3:

MCF7, grown in serum-free media overnight, then treated with EGF 1μg/ml for 10min whole cell lysate, The membrane was incubated with phosphatase at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 55 kDa

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Exposure time: 1min

• WB

Lab

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

All lanes:

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (ab40795) at 1/2000 dilution

Lane 1:

HeLa cell lysate with None

Lane 2:

HeLa cell lysate with PAK2 (pS141)

Lane 3:

HeLa cell lysate with PAK2 non-phospho

Lane 4:

HeLa cell lysate with PAK3 (pS154)

Lane 5:

HeLa cell lysate with PAK3 non-phospho

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• WB

Lab

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

All lanes:

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (ab40795) at 1/10000 dilution

Lane 1:

HeLa whole cell lysate - untreated at 10 µg

Lane 2:

HeLa whole cell lysate - treated with phosphatase at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

• WB

Lab

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

All lanes:

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (ab40795) at 1/10000 dilution

Lane 1:

RAW264.7 (mouse abelson murine leukemia virus-induced tumor) whole cell lysate - treated with phosphatase at 10 µg

Lane 2:

RAW264.7 (mouse abelson murine leukemia virus-induced tumor) whole cell lysate - untreated at 10 µg

Secondary

All lanes:

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

false

• WB

Lab

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

All lanes:

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (ab40795) at 1/50000 dilution

Lane 1:

C6 (rat glioma) whole cell lysate - treated with phosphatase at 10 µg

Lane 2:

C6 (rat glioma) whole cell lysate - untreated at 10 µg

Secondary

All lanes:

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

false

• WB

CiteAb

Western blot - Anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] (AB40795)

PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) western blot using anti-PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) antibody [EP656Y] ab40795. Publication image and figure legend from Schelle, I., Bruening, J., et al., 2016, Toxins (Basel), PubMed 28025502.

ab40795 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab40795 please see the product overview.

Effects of genetic deletion of p38alpha and of SB203580 treatment on TcsL-catalyzed Rac/Cdc42 glucosylation (time-dependency). p38alpha-/- MSCV p38alpha MEFs and p38alpha-/- MSCV empty vector (EV) MEFs were treated with TcsL (1 μg/mL) in the presence of SB203580 (10 μM) or DMSO alone for the indicated times. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, PAK2, pT222-MAPKAPK2, MAPKAPK2, and beta-actin were analyzed by immunoblot using the indicated antibodies. Quantifications of immunoblots were performed using Kodak software and relative amounts of non-glucosylated Rac/Cdc42 versus the total levels of Rac1, respectively, are expressed as mean ± SD of three independent experiments. * indicates significant differences, p < 0.05, as analyzed using Student's t-test.

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