Skip to main content
. 2014 Jan 9;10(1):e1003891. doi: 10.1371/journal.ppat.1003891

Figure 1. Chk2 but not SMC1 was activated during MVM infection.

Figure 1

(A) Time course of Chk2 activation following MVM infection of murine A9 cells. A9 cells were para-synchronized in G0 as described in Materials and Methods. Cells were then mock-infected or infected with MVMp at an MOI of 10 at the time of release (T0, lane 1). As a positive control for Chk2 activation, A9 cells were treated with 150 ng/ml of the radiomimetic neocarzinostatin (NCS, lane 7) for 1 hour. Treatment with calf intestinal phosphatase (CIP, lane 6) was done for 1 hour at 37°C. Cells were harvested at the indicated time points after release, lysed in modified RIPA buffer. Protein content was measured using Bradford assay and equal amounts of protein were loaded in each well for immunoblotting. Western blot analysis was carried out using antibodies directed against NS1 (panel a), Chk2 (panel c) and H2AX phosphorylated on serine 139 (γH2AX, panel d). Equal loading was confirmed by blotting for actin (panel b). (B) Chk2 activation following MVM infection in permissive human NB324K cells. NB324K cells were mock infected or infected with MVM at an MOI of 10. Cells were harvested 24 hours post infection. Western blot analysis using antibodies directed against NS1, Actin, Chk2 phosphorylated on threonine 68 (Chk2-P-T68) and total Chk2 protein (Chk2) as indicated is shown. (C) Activated Chk2 localized within MVM APAR bodies. NB324K cells were infected for 24 hours before fixation and processing for immunofluorescence. APAR bodies were detected with antibodies to NS1. Nuclei were stained with TOPRO-3. Staining with an antibody to phosphorylated Chk2, observed only in infected cells, was prominent in distinct foci which co-localized with APAR bodies and also in a pan-nuclear pattern (merge panel). All images were captured using an objective of 63×. (D) SMC1 is not significantly activated following MVM infection of murine A9 cells. Lysates from Figure 1A were blotted with antibodies directed against total SMC1 protein (SMC1) and SMC1 protein phosphorylated on serine 957 (SMC1-P-S957). Short and long exposures of the same blot are shown.