Anti-PAK4 (phospho S474) + PAK5 (phospho S474) + PAK6 (phospho S474) Antibody [RP23040229]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAK4 (phospho S474) + PAK5 (phospho S474) + PAK6 (phospho S474) Antibody [RP23040229] (AB313491)

Immunofluorescent analysis of MCF-7 cells fixed with 4% formaldehyde reconstituted in 1X PBS for 10 min at room temperature and permabilized using 0.1 % Triton X-100 in PBS for 15 min at room temperature labeling endogenous Phospho-PAK4 + PAK6 + PAK7 pSer474 with ab313491 at 2 µg/mL dilution followed by Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate at 1/2000 dilution. Panel a) shows representative cells that were stained for detection and localization of Phospho-PAK4 + PAK6 + PAK7 pSer474 protein (green), Panel b) is stained for nuclei (blue) using DAPI. Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin at 1/300 dilution. Panel d) is a composite image of panels a, b and c clearly demonstrating nuclear localization of Phospho-PAK4 + PAK6 + PAK7 pSer474. Panel e) shows loss of signal by competition with the Phospho-PAK4 + PAK6 + PAK7 pSer474 phospho peptide, demonstrating antibody specificity. Panel f) demonstrates no competition with the non-phosphorylated peptide. Panel g) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

• WB

Supplier Data

Western blot - Anti-PAK4 (phospho S474) + PAK5 (phospho S474) + PAK6 (phospho S474) Antibody [RP23040229] (AB313491)

All lanes:

Western blot - Anti-PAK4 (phospho S474) + PAK5 (phospho S474) + PAK6 (phospho S474) Antibody [RP23040229] (ab313491) at 1 µg/mL

Lane 1:

Hela cell lysate at 30 µg

Lane 2:

A549 cell lysate at 30 µg

Lane 3:

MCF7 cell lysate at 30 µg

Lane 4:

phosphopeptide (10 µg/ml) incubated Hela cell lysate at 30 µg

Lane 5:

phosphopeptide (10 µg/ml) incubated A549 cell lysate at 30 µg

Lane 6:

phosphopeptide (10 µg/ml) incubated MCF7 cell lysate at 30 µg

Lane 7:

non-phosphopeptide (10 µg/ml) incubated Hela cell lysate at 30 µg

Lane 8:

non-phosphopeptide (10 µg/ml) incubated A549 cell lysate at 30 µg

Lane 9:

non-phosphopeptide (10 µg/ml) incubated MCF7 cell lysate at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate at 1/5000 dilution

Predicted band size: 64.072 kDa

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