P pak4

Manufactured by Cell Signaling Technology

Sourced in United States

P-PAK4 is a monoclonal antibody that detects endogenous levels of PAK4 protein when phosphorylated at serine 445.

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P-Pak4 Ser474 (2 citations) Rabbit anti-p-Pak4 Ser474 (2 citations)

4 protocols using p pak4

Protein Expression Quantification Protocol

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MTT solution, mouse monoclonal anti-β-actin, NA, NMN and NAD were obtained from Sigma (St. Louis, MO, USA). The antibodies against (PAK4, p-PAK4, β-catenin, p-β-catenin, cycline D1, c-Myc, β-actin (rabbit), PARP, Sirtuin 1) were from cell signaling Technology, Inc. (Beverly, MA, USA). Goat anti-mouse and goat anti-rabbit HRP conjugated IgG were obtained from Bio-Rad (Hercules, CA). Anti-NAMPT was from Bethyl Laboratories (Montgomery, TX, USA), anti-NAPRT1 was from Proteintech (Rosemont, IL 60018, USA). ECL Plus solution was from Thermo-Fisher Scientific (Waltham MA, USA). KPT-9274 and its vehicle were from Karyopharm Therapeutics (Newton, MA, USA). FK866 was from TOCRIS Biosciences. Sunitinib was obtained from LC laboratories (Woburn, MA).

Aboud O.A., Chen C.H., Senapedis W., Baloglu E., Argueta C, & Weiss R.H. (2016). Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth. Molecular cancer therapeutics, 15(9), 2119-2129.

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Protein Profiling in Nuclear and Cytoplasmic Fractions

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Nuclear and cytoplasmic fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, Waltham, MA, USA). Tissue homogenates or cell lysates (15 μg) were separated by 6–14% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, blots were probed with primary antibodies against: PAK4 (G222), p-PAK4 (S474), p-IKKβ (S176/180), p-IκBα (S32), p-p65 (S536), cleaved caspase-3 (Asp175), Bax (Cell Signalling Technology, Beverly, MA, USA), GAPDH (A531), lamin B1(L75), Bcl2(P65), NQO1 (F252) (Bioworld Technology, St Louis Park, MN, USA), NF-κB p65 (C-20), IKBα (H-4), IKKβ (10A9B6) (Santa Cruz Biotechnology, Dallas, TX, USA), Nrf2 (Proteintech, Rosemont, IL, USA), HO-1 (Enzo Life Sciences, Farmingdale, New York, USA), and p-Thr (Merck KGaA, Darmstadt, Germany). For co-immunoprecipitations, 600 μg protein was incubated with anti-Nrf2 antibodies (Proteintech, Sankt Leon-Rot, Germany) overnight at 4 °C followed by protein G agarose for 2 h at 4 °C. Blots were probed with antibodies against PAK4, Nrf2, or p-Thr. Immunoreactive bands were detected with a Las-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).

Mao Y., Lee E., Yang X., Bae E.J., Jeon R, & Park B.H. (2022). Targeting p21-activated kinase 4 (PAK4) with pyrazolo[3,4-d]pyrimidine derivative SPA7012 attenuates hepatic ischaemia-reperfusion injury in mice. Journal of Enzyme Inhibition and Medicinal Chemistry, 37(1), 2133-2146.

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Pancreatic Cancer Cell Characterization

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PDAC cells (MiaPaCa-2, HPAC and Panc1) were purchased from ATCC. Colo-357 and L3.6pl cells were provided by Dr. Paul J Chiao (MD Anderson Texas). HPDE cells were generously provided by Dr. Mien-Sound Tsao (Department of Pathology, Montreal General Hospital, Quebec). The cells were grown in DMEM culture medium (Invitrogen) complemented with 10% FBS (Cambrex), 100 U/ml penicillin-streptomycin (Invitrogen), 2 mM L glutamine (Invitrogen). Cell lines used in this study were obtained in 2009. MiaPaCa-2, HPAC and Panc1 have been authenticated yearly and recently on July 14^th 2016. The method used for testing was short tandem repeat (STR) profiling using the PowerPlex® 16 System at Genetica® (Burlington, NC USA). MiaPaCa-2 CSCs were developed by flow sorting authenticated MiaPaCa-2 cells for CD44 CD133 and EpCAM. These triple positive cells were grown as spheroids in 1:1 DMEM/F12 medium supplemented with B-27 and N-2 (Invitrogen) in ultra-low attachment six well plates (Corning, USA). These cells sustained in long term culture conditions and show stem cell characteristics with mesenchymal markers (12 (link), 13 (link)). PAMs were provided by Karyopharm Therapeutics (Newton MA, USA). Primary antibodies for PAK4, p-PAK4, GEFH1, Cyclin D1, ERK1/2, Vimentin and p65 were purchased from Cell Signaling (Danvers, MA). All the secondary antibodies were obtained from Sigma (St. Louis, MO, USA).

Aboukameel A., Muqbil I., Senapedis W., Baloglu E., Landesman Y., Shacham S., Kauffman M., Philip P.A., Mohammad R.M, & Azmi A.S. (2016). Novel p21-activated kinase 4 (PAK4) allosteric modulators overcome drug resistance and stemness in pancreatic ductal adenocarcinoma. Molecular cancer therapeutics, 16(1), 76-87.

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Protein Quantification and Western Blot Analysis

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Total protein was extracted from cells using ice-cold radioimmunoprecipitation assay buffer supplemented with phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). ABCA kit with Varioskan multimode microplates spectrophotometer from Thermo Fisher Scientific Inc. (Waltham, MA, USA) was used to detect the concentration of protein in the supernatant. A total of 20 µg protein/well was fractionated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membranes were blocked at room temperature for 2 h with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated with P-ERK (1:1,000; Cell Signaling Technology; cat. no. 9101), P-Pak1 (1:1,000; Thermo Fisher Scientific; cat. no. PA5-37677), P-Pak4 (1:1,000; Cell Signaling Technology; cat. no. 3241) and P-AKT (1:1,000; Cell Signaling Technology; cat. no. 4060) at 4°C overnight. The membranes were washed in TBST and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Goat anti-rabbit, 1:5,000; ZSGQ-BIO; cat. no. ZF-0311) for 2 h at room temperature. Bound antibody complexes were detected and visualized by ECL western bolting detection kit (Ambion; Thermo Fisher Scientific, Inc.).

Xiao F., Cheng Z., Wang P., Gong B., Huang H., Xing Y, & Liu F. (2018). MicroRNA-28-5p inhibits the migration and invasion of gastric cancer cells by suppressing AKT phosphorylation. Oncology Letters, 15(6), 9777-9785.

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