Human phospho kinase antibody array

Total cellular extracts were performed as previously described in Van Seuningen et al. [34 (link)] and Jonckheere et al. [35 (link)]. Western-blotting on nitrocellulose membrane (0.2 µm, Whatman, Maidstone, UK) was carried out as previously described [36 (link)]. Membranes were incubated with antibodies against STAT3 (79D7, Cell signalling), phospho S727 STAT3 (9134, signalling), c-Jun (60A8, Cell signalling), phospho S63 c-Jun (54B3, Cell signalling) and β-actin (AC-15, Sigma, Tokyo, Japan). Antibodies were diluted in 5% (w/v) non-fat dry milk in Tris-Buffered Saline Tween-20 (TBS-T). Peroxydase-conjugated secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) were used and immunoreactive bands were visualised using the West Pico chemoluminescent substrate (Thermo Scientific, Pierce, Brebières, France). Intracellular signaling was studied using Human Phospho-Kinase Antibody Array (ARY003B, R & D) (detecting relative site-specific phosphorylation of 43 proteins) and Human Apoptosis Array Kit (ARY009, R & D). Chemo-luminescence was visualised using LAS4000 apparatus (Fujifilm, Tokyo, Japan). Density of bands were integrated using Gel analyst software^® (Claravision, Paris, France) and represented as histograms. Three independent experiments were performed.

Human Phospho-Kinase Antibody Array (RD Systems^® [Minneapolis, Minnesota]) was used to analyze the relative site-specific phosphorylation of 43 kinases and 2 related total proteins. Following the manufacturer’s instructions, we used 1.107 cells/mL for cell lysis corresponding to 400 µg of protein per array [43 (link)]. Phosphorylated proteins were detected using Syngene PXi by exposing membranes to luminescent signals generated after incubation of the membrane with ECL (WBKLS0500, Millipore^® [Darmstadt, Germany]). Image J^® software (National Institutes of Health and Laboratory for Optical and Computational Instrumentation [University of Wisconsin, Madison, WI, USA]) was used to quantify dot intensity.

Samples for western blot were prepared, runned and immunostained as described in [55 (link)]. Human L507 array (Raybiotech, Norcross, GA) was used to detect relative levels of 507 proteins in CM^CAAT. Human phospho-kinase antibody array (R&D systems) was used to detect relative phosphorylation levels of 44 kinases. Concentrations of human leptin, adiponectin, IL-6, C-C motif chemokine 22 (CCL22) and colony stimulating factor 1 (CSF-1) in CM^CAAT were measured with a Duoset or Quantikine ELISA Kit (R&D systems). Scanning densitometry was carried out with the Quantity One Program (Bio-Rad, Hercules, CA). Functional analysis of detected proteins was performed using FunRich version 2.1 [56 (link)]. Venn diagrams were created with BioVenn software (www.cmbi.ru.nl/cdd/biovenn/).

Lysates for western blotting and immunoprecipitation were prepared using the normal lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100) containing protease inhibitor cocktail (p8340, Sigma) and 0.2 mM sodium orthovanadate. Immunoprecipitation was performed using protein-A/G agarose beads (Invitrogen), as previously described [27 (link)]. SDS-PAGE electrophoresis and western blotting were performed using the NuPAGE SDS PAGE Gel System and NuPAGE Bis Tris Precast Gels (4–12%) (Life Technologies). Western Lightning PLUS Enhanced Chemiluminescent Substrate (PerkinElmer) was for imaging western blots on the Vilber Lourmat Fusion chemiluminescent imaging system. Quantitative western blotting was performed using multistrip western blotting [28 (link)]. The Human Phospho-Kinase Antibody Array was obtained from R&D Systems (MN, USA) and used according to manufacturer’s instructions.

Human Phospho-Kinase Antibody Array (R&D Systems) was performed as per the manufacturer’s instruction. Briefly, the cell lysates (800 μg) were mixed with array buffer and incubated with pre-blocked array membrane at 4 ^oC for overnight. Membranes were then washed and incubated with primary antibody cocktail for 2 h, followed by washing and incubated with secondary antibody for 30 min. Membranes were washed again and subjected to chemiluminescent detection.