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aFunctional modules in motile cilia encompass at least 57 proteins, and include subunits of the Inner Dynein Arm (IDA) and Outer Dynein Arm (ODA), which confer motility, as well as subunits within radial spokes (RS) and the Nexin-Dynein Regulatory Complex (N-DRC), which regulate motility. ...
Total RNA was extracted using phenol–chloroform extraction using TRIzol reagent (Invitrogen). Reverse transcription was performed with a First Strand cDNA synthesis kit (Roche) following the manufacturer’s instructions with random hexamers. Real-time PCR was performed with GoTaq qPCR Master Mix (Prom...
PDA possesses multiple phenol groups, which can donate protons or electrons to stabilize free radicals, including extracellular ROS [30]. In this study, it has been observed that BG-PDA effectively decreased intracellular ROS levels, as determined by flow cytometry analysis. Remarkably, the results ...
To remove genomic DNA contamination RNA preparations were treated with RNase-free DNase I (Qiagen), followed by phenol/chloroform extraction and ethanol precipitation. Photometric analyses of all RNA samples showed A260/A280 ratios higher than 2.1. Two micrograms of each total RNA sample were used ...
Extraction of total RNA was performed using a modified hot phenol method [28]. Purification of RNA and DNA digestion with DNAse was performed using the commercial kit Nucleospin ® RNA (Macherey-Nagel, Germany). Synthesis of cDNA from total RNA samples was performed using the MultiscribeTM ...
Phenol oxidase Here we refer to PO activities instead of a single PO enzyme to best reflect the biochemical understanding of this enzyme group (Supplementary Note 1). We measured PO activity through the l-3,4,-dihydroxy phenylalanine (l-DOPA) assay (see ‘Enzyme assays’). To survey PO gen...
To identify the enzyme(s) responsible for increased 9-FAHFA biosynthesis in AG4OX adipocytes, we performed activity-based protein profiling using a fluorophosphonate (FP)-alkyne probe23,25that uses the same functional group as MAFP and therefore should inhibit the same enzymes. Covalent labelling ...
and incubated at 37°C overnight. The digested solution was extracted progressively with phenol, phenol and chloroform mixture, and chloroform, and DNA was precipitated with ethanol at a final concentration of 65%. The DNA was further purified and dissolved in 2.0 mM Tris (pH 8.0). The quanti...
Phenol-chloroform extraction of isolated chromatin prior to immunoprecipitation abolished the PCR product in all samples (Figure 6C, lanes 7 and 8), validating that the immunoprecipitation requires the presence of protein bound to DNA. This ChIP demonstrates that Nub protein physically interacts with ...