Kinase activity was measured using protein tyrosine kinase (PTK) or serine-threonine kinases (STK) PamChip4 porous 3D microarrays and measured using the PamStation12 (PamGene International, ’s-Hertogenbosch, The Netherlands). Substrates contained in each array are included in the project GitHub repository listed above. Mouse livers were pooled and measured in triplicate across three chips simultaneously for PTK and STK, as previously described in [50 (link)]. This approach effectively deals with large batch effects across samples. It allows for the characterization of kinase activity only in the context of analytical variance. The pooled samples were lysed using M-PER Mammalian Extraction Buffer (Thermo Fischer Scientific, CAT#78503), Halt Phosphatase Inhibitor (Thermo Fischer Scientific, CAT#78428), and Protease Inhibitor Cocktail (Sigma, CAT#P2714). The samples were homogenized using TissueLyser LT (Qiagen). The protein concentration was measured in triplicate using Pierce BCA Protein assay (Thermo Fischer Scientific, CAT#23225). Samples were diluted to a final protein concentration of 2.5 μg/μL. Each array contained 1 μg of protein per sample for STK chips and 5 μg for PTK chips. In the presence of adenosine triphosphate (ATP), kinase phosphorylation activity is quantified using fluorescently labeled antibodies to detect differential phosphorylation of 196 (PTK) or 144 (STK) reporter peptides between experimental and control conditions, as previously described [50 (link)]. Evolve (PamGene) software uses a charge-coupled device (CCD) camera and light-emitting diode (LED) imaging system to record relative phosphorylation levels of each unique consensus phosphopeptide sequence every 5 min for 60 min as measured by peptide signal intensities recorded across 10, 20, 50, and 100 millisecond exposure times. Raw imaging data were exported for further data analysis and kinase mapping.
Free full text: Click here
