Fig 1: CK2 activity is important for viral replication and for regulating VIB morphology during BTV infection. HeLa cells infected with BTV1 (MOI = 1) were treated 4 h.p.i. with concentrations of 100, 50, 20, and 10 μm TBB for 20 h. A, samples were analyzed by Western blotting using specific antibodies as indicated. B, densitometry analysis of the Western blots expressed as a percentage. C, virus titer derived from HeLa cells infected with BTV1 (MOI = 1) and treated 4 h.p.i. with concentrations of 100, 50, 20, and 10 μm TBB for 20 h. D, HeLa cells infected with BTV1 (MOI = 1) were treated 20 h.p.i. with concentrations of 100, 50, 20, and 10 μm TBB for 4 h. E, VIBs denoted by NS2 staining were quantified using Volocity software, and their size was determined in replicate experiments of D. Error bars represent the standard deviation (Western blots) and standard error (confocal images) values of stimulations from three independent experiments. *, p < 0.05.
Fig 2: CK2 K198R exhibits altered +1 substrate specificity. Substrate specificity of CK2WT (A–C) and CK2K198R (D–F). The CK2 K198R mutation results in a decreased preference for acidic (D/E) residues in the +1 position for S and T centered phosphorylation sites. For Y centered phosphorylation sites, the preference for acidic residues is shifted to the –2 and –1 positions.
Fig 3: CK2K198R mutation shifts overall specificity motif. Substrate specificity of CK2WT (A) and CK2K198R (B). The CK2 K198R mutation results in a decreased preference for acidic (D/E) residues in the +1 position as well as a reordering of phosphoacceptor preference. In CK2K198R, a decreased preference for threonine is paired with an increase in preference for tyrosine.
Fig 4: CK2 K198R phosphorylates an expanded complement of substrates. Venn diagram representing the overlap of phosphorylation sites identified in CK2WT and CK2K198R ProPeL experiments (A). Motifs of phosphopeptides identified only in CK2WT (B), both CK2WT and CK2K198R (C) or only in CK2K198R (D) ProPeL experiments. Phosphopeptides identified in both CK2WT and CK2K198R experiments tend to reflect the canonical CK2WT motif. Those identified only in CK2K198R experiments have a much wider complement of amino acids in the +1 position, indicating increased promiscuity in this position. The phosphoacceptor abundance is also altered, reflecting an increased preference for tyrosine in the K198R mutant.
Fig 5: NS2 phosphorylation at Ser-249 and Ser-259 decreases following CK2 inhibition or PP2A activation. HeLa cells infected with BTV1 (MOI = 1) were treated 20 h.p.i. for 4 h with 100 μm TBB, 10 μm FTY720, and 20 nm okadaic acid. NS2 was isolated from these cells using immunoprecipitation (IP). A, samples were analyzed by Western blotting to confirm NS2 identity. SDS-PAGE gels were sequentially stained for phosphoproteins and total proteins. B, densitometry analysis of phosphoprotein and total protein staining data in A from replicate experiments. NS2 is indicated along the detected Ig heavy chains (HC). Error bars represent the standard deviation values of stimulations from three independent experiments. *, p < 0.05.
Supplier Page from Abcam for Anti-CSNK2A1 antibody