Proteome profiler human phospho kinase array

HepG2 cells were incubated in DMEM containing 5.5, 25, and 25 mM glucose + hrANXA1 (20 μg/mL) or 25 mM glucose + hrANXA1 (20 μg/mL)+ WRW4 (0.9 mM) and incubated for 48 h. Cells were washed in PBS containing protease inhibitors and cell lysates extracted as per manufactures instructions. Protein concentration was quantified and 600 μg of protein was used per membrane of a Proteome Profiler, Human Phospho-kinase Array (R&D Systems). After visualization of the spots, the signals were measured using Image Studio LICOR, normalization was carried as per manufacturers instruction and changes plotted in a heat map expressed as fold change to 5.5 mM glucose for each individual phosphorylation site using PRISM.

The proteome profiler human phospho-kinase array (R&D systems, ARY003B, MN, USA) was used to determine the phosphorylated protein profile. Whole-cell extracts were collected and incubated in the arrays of human phospho-kinase antibodies. Phosphorylation status was measured by incubation with anti-phosphotyrosine horseradish peroxidase following protocols.

The phosphorylation profile was analyzed in the cells using the Proteome Profiler Human Phospho-Kinase Array (R&D Systems). Cells (1 × 10⁴/ml) were plated in DMAM-10% FBS in a 100-mm dish and grown for 72 h to produce a subconfluent culture. Cell lysate samples (0.5 mg; 1.5 mg/ml each) were applied per array set comprised of two nitrocellulose membranes with the spotted capture antibodies. The bound material was detected using the biotinylated antibodies followed by streptavidin conjugated with horseradish peroxidase. The chemiluminescent signal was acquired using the HyBlot CL autoradiography film (Denville, South Plainfield, NJ, USA). The film was scanned and digitized. Pixel density of the spots was quantified using the ImageJ software.

Total protein extraction and purification were performed from cell lysates as previously described^{20 (link)}. Briefly, cells were lysed for 5 min on ice in 200 μl of ice-cold RIPA buffer (65 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.5% sodium deoxycholate) supplemented with phosphatase inhibitor cocktails I and II and a protease inhibitor cocktail (Sigma, Darmstadt, Germany). Then, standardized concentration of total proteins was subjected to a Proteome Profiler Human Phospho-Kinase Array (R&D Systems, Lille, France) following manufacturer’s instructions. The density of spots, corresponding to protein activation, was measured by MyImage^TM Analysis Software 2.0 (Thermofisher) for each molecule and each condition.

For the Proteome Profiler Human Phospho-Kinase Array (R&D, Abingdon, UK) 1 × 10⁶ primary PCa cells were seeded in a collagen I-coated 10 cm-well plate and treated with 48 h-conditioned media from STO-IL-4 or STO-GUS for 30 min or 48 h. The array was performed according to the manufacturer’s manual and developed in a GeneGnome XRQ imaging system (Syngene, Cambridge, UK).

Considering the established role of SPRY2 in RTK signalling [11 (link), 25 (link)], we hypothesised that key cellular signalling pathways may be affected in these cells. To investigate this, we performed a phospho-kinase array for the simultaneous determination of phosphorylation levels in 43 protein kinases across multiple pathways relevant to metabolism and insulin resistance (including Akt, MAPK, mTOR and Jak/STAT signalling). This was carried out using the Proteome Profiler Human Phospho-Kinase Array (R&D Systems) following the manufacturer’s protocol for LI-COR near-infrared fluorescence detection. Cells were incubated in serum-free medium with or without insulin (100 nM for 10 min [19 (link)]) then lysed in Lysis Buffer 6 from the array kit. Protein concentration was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher). Total cellular proteins (300 μg) were hybridised to the array membranes overnight. The arrays were washed and incubated with the detection antibody cocktails for 2 h, followed by IRDye 800CW Streptavidin (LI-COR 925–32,230; 1:2000) for 30 min. Membranes were imaged on the LI-COR Odyssey infrared imaging system and density of spots determined using Fiji [26 (link)].

Proteome Profiler Human Phospho-Kinase Array (R&D Systems) were incubated with 400 μg lysate overnight at 4 °C. The following day, chemiluminescent detection was performed according to manufacturer's indications. Densitometry analysis was conducted using ImageJ.