BGC 823 cells were incubated with indicated concentration of small molecular compounds. Then cells were suspended in cold lysis buffer supplemented with protease and phosphatase inhibitors. Supernatants were preabsorbed with protein A-Sepharose 4B beads (GE Healthcare Bio-Science Inc., Sweden) for 1 h at 4 °C before incubation with PAK1 antibody (Santa Cruz). Then the supernatants with equal amounts of protein were subjected to immunoprecipitation with PAK1 antibody. MBP was used as substrates to assess PAK1 kinase activity. Kinase activity was measured in kinase buffer containing 10 μCi of [r-32P] ATP (5000 Ci mmol−1) for 30 min at 30 °C. Reactions were terminated by addition of 6 × SDS buffer and loading on a 12% SDS-PAGE. 32P-labelled proteins were transferred onto PVDF membranes and detected by autoradiography with Molecular Imager RX (BIO-RAD).
Pak1 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in Sweden
The PAK1 antibody is a research tool used to detect and study the p21-activated kinase 1 (PAK1) protein. PAK1 is a serine/threonine protein kinase that plays a role in various cellular processes. The antibody can be utilized in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to identify and analyze the PAK1 protein in biological samples.
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Lab products found in correlation
Molecular imager rx, by Bio-Rad (1 mentions)
Alexa fluor 488 anti chicken, by Thermo Fisher Scientific (1 mentions)
Phospho ser144 pak antibody, by Cell Signaling Technology (1 mentions)
Hoechst, by Merck Group (1 mentions)
Anti map2, by Santa Cruz Biotechnology (1 mentions)
Chicken anti gfp, by Aves Labs (1 mentions)
Anti mouse alexa fluor 633, by Thermo Fisher Scientific (1 mentions)
Rac1 antibody, by BD (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
2 protocols using pak1 antibody
1
Assessing PAK1 Kinase Activity
2
Phospho-PAK Quantification in Cell Lines
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List of all primary antibodies used: for immunoblotting, phospho-Ser144 PAK antibody (rabbit; Cell Signaling, #2606S, 1/1000), PAK1 antibody (mouse; Santa Cruz sc-166887, 1/1000), GFP antibody (rabbit; Torrey Pines Biolabs, #TP401, 1/3000), RAC1 antibody (mouse; BD Biosciences 610651, 1/1000); for immunocytochemistry, anti-GFP (chicken, Aves Labs, 1/1000), anti-MAP-2 (Santa Cruz, 1/500). List of all secondary antibodies used: Dylight Rabbit 680 (Thermo Fisher Scientific, 35568), Dylight Mouse 800 (Thermo Fisher Scientific, SA5-35521), Alexa Fluor 488 anti-chicken (Thermo Fisher Scientific, A11039), Alexa Fluor 633 anti-mouse (Thermo Fisher Scientific, A21050), Phalloidin TRITC (Sigma-Aldrich, P1951, 1/40,000), Hoechst (Sigma-Aldrich, B2261, 1/50,000).
Cell culture, transfection and immunoblot analysis of Phospho-PAK amounts HEK293T and N1E-115 neuroblastoma cell lines were obtained from ATCC and were regularly tested for mycoplasma contamination by PCR. Cells were cultured and transfected as described in [25] . Immunoblot analysis for quantification of Phospho-PAK levels were also performed as described in [25] , as was the quantification of lamellipodia in N1E-115 cells.
Cell culture, transfection and immunoblot analysis of Phospho-PAK amounts HEK293T and N1E-115 neuroblastoma cell lines were obtained from ATCC and were regularly tested for mycoplasma contamination by PCR. Cells were cultured and transfected as described in [25] . Immunoblot analysis for quantification of Phospho-PAK levels were also performed as described in [25] , as was the quantification of lamellipodia in N1E-115 cells.
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