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M1 Y132 is phosphorylated by JAK2 at late stages of virus replication.

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posted on 31.08.2020, 17:38 by Angeles Mecate-Zambrano, Swathi Sukumar, Guiscard Seebohm, Kevin Ciminski, André Schreiber, Darisuren Anhlan, Lilo Greune, Ludmilla Wixler, Stephanie Grothe, Nora Caroline Stein, M. Alexander Schmidt, Klaus Langer, Martin Schwemmle, Tianlai Shi, Stephan Ludwig, Yvonne Boergeling

A) Mass spectrometry was performed on overexpressed GST-M1 isolated from WSN-infected A549 cells (MOI 5; 7 and 9 hpi). Depicted are the gas phase fragment ions detected for phosphorylated and carbamidomethylated peptide REITFHGAKEIALSYSAGALACCMGLIYNR (m/z 3452.631) in data-independent mass spectrometry (MSe) using Synapt G2 Si. B) Plaque sizes of WSN WT, M1 Y10F and M1 Y132A were quantified from neutral red-stained dilutions of standard plaque assays by using Adobe Photoshop ruler tool. Plaques (n = 30) were randomly selected and measured in diameter. Results are depicted as relative plaque size compared to WT ±SD. Statistical significance was analyzed by one-way Anova followed by Dunn’s multiple comparisons test. C) Quantification of viral protein expression in WSN WT versus M1 Y10F infection (see Fig 1E). Densitometric analyses of band intensities were performed by using Image Studio version 5.2 and are depicted as n-fold expression ±SD of three independent experiments. D) in vitro phosphorylation of recombinant M1 WT, M1 Y132A, M1 Y132F or GST alone by recombinant JAK2. Tyrosine phosphorylation was analyzed by Western blot. Blots are representative of two independent experiments.