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Phospho-PAK4 + PAK5 + PAK6 pSer474 Antibody (711694) in WB
Western blot analysis was performed on Whole cell extracts (30 ug lysate) of HeLa (Lane 1), A549 (Lane 2) and MCF-7 (Lane 3). The blots were probed either, with ABfinity™ Anti-Phospho-PAK4 + PAK5 + PAK6 [pSer474] Rabbit Oligoclonal Antibody (Product ## 711694,1 ug/ml). To confirm the specificity competition was performed with the phosphopeptide (10 µg/mL) (Lane 4, 5, 6) and non phosphopeptide (10 µg/mL) (Lane 7, 8, 9). A 72 kDa band corresponding to Phospho-PAK4 + PAK5 + PAK6 [pSer474] was observed across the cell lines tested, which specifically gets competed with phosphopeptide and not with the nonphosphorylated peptide. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product ## A27036, 0.4 ug/ml, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product ## NP0321BOX), XCell SureLock™ Electrophoresis System (Product ## EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product ## LC5800).Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product ## IB21001).The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product ## WP20005)
![Western blot analysis was performed on Whole cell extracts (30 ug lysate) of HeLa (Lane 1), A549 (Lane 2) and MCF-7 (Lane 3). The blots were probed either, with ABfinity™ Anti-Phospho-PAK4 + PAK5 + PAK6 [pSer474] Rabbit Oligoclonal Antibody (Product ## 711694,1 ug/ml). To confirm the specificity competition was performed with the phosphopeptide (10 µg/mL) (Lane 4, 5, 6) and non phosphopeptide (10 µg/mL) (Lane 7, 8, 9). A 72 kDa band corresponding to Phospho-PAK4 + PAK5 + PAK6 [pSer474] was observed across the cell lines tested, which specifically gets competed with phosphopeptide and not with the nonphosphorylated peptide. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product ## A27036, 0.4 ug/ml, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product ## NP0321BOX), XCell SureLock™ Electrophoresis System (Product ## EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product ## LC5800).Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product ## IB21001).The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product ## WP20005)](http://tools.thermofisher.com/content/sfs/gallery/low/711694-Phospho-PAK4PAK5PAK6pSer474-WB_20170512081144.jpg)
