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. 2020 Sep 22;48(18):10428–10440. doi: 10.1093/nar/gkaa771

Figure 5.

Figure 5.

ERI1 is co-purified with histone mRNA and SLBP in infected cells. (A–C) HEK-293T cells were transfected with Strep-ERI1 wild-type (wt), Strep-ERI1 R105A mutant, Strep-ERI1 ΔSAP mutant or with Strep-mCherry or Strep-empty (as a control), and infected with WSN (3pfu/cell). At 6hpi, Strep-tagged proteins were purified with sepharose Strep-Tactin beads. The levels of HIST1H2AB mRNA (A) and NP mRNAs (B) co-purified with Strep-pulled proteins are expressed as the mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA test with Dunnett's multiple comparison test (****P < 0.0001). (C) Inputs and Strep-pulled eluates were analyzed by immunoblot with the indicated antibodies. (D) HEK-293T cells were co-transfected with Strep-ERI1 and 3XFlag-SLBP, or transfected with either Strep-ERI1 or 3XFlag-SLBP, and infected with WSN (moi = 3 pfu/cell). At 6 hpi, PB2 proteins were purified using anti PB2 antibodies (or control immunoglobulins IgG ctrl). Inputs and eluates were analyzed by immunoblot using Strep-Tactin or antibodies directed against 3X-Flag or PB2 to respectively detect Strep-ERI1, 3XFlag-SLBP and PB2. (E) HEK-293T cells were transfected with Strep-ERI1, Strep-SLBP or Strep-mCherry and infected with WSN (moi = 3pfu/cell). At 6hpi, Strep tagged proteins were purified with sepharose Strep-Tactin beads. Complexes bound to the beads were incubated with RNase A (+) or Rnasin (−) for 1h. Inputs and eluates were analyzed by immunoblot to detect Strep, PB2 and NP. Results representative of three independent experiments are shown. (F) HEK-293T cells were infected with WSN at an moi of 3 pfu/cell. At 6hpi, PB2 proteins were purified using anti-PB2 antibodies. Levels of HIST1H2AB, HIST1H2BG and HIST1H3B mRNA co-purified with PB2 (black) or with control IgG (gray) were quantified using RT-qPCRs. The results are expressed as the mean ± SEM of triplicates and the significance was tested with a multiple t test, using the Holm-Sidak method, in GraphPad Prism software (*** P < 0.001, **** P < 0.0001). (G) HEK-293T cells were treated with NT (dark grey) or ERI1 (light gray) siRNAs. At 48hpt, cells were infected with WSN (moi = 3 pfu/cell). At 6hpi, PB2 proteins were purified using anti-PB2 antibodies. Levels of HIST1H2AB, HIST1H2BG and HIST1H3B mRNA co-purified with PB2 in NT and ERI1 siRNA treated cells were quantified using RT-qPCRs. The results are expressed as the mean ± SEM of triplicates.

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