Fig. 3.

The region between residues 240 and 482 of the CCHFV N protein is important for N–N interactions. (a) Denaturation of CCHFV N protein. HEK 293T cells were transfected with Flag–N, as indicated in Methods. At 48 h post-transfection, cells were collected in lysis buffer containing protease inhibitors and cell lysates were clarified at 16 000 g for 20 min at 4 °C. Aliquots of the cytoplasmic extracts were treated with RNase A at a final concentration of 0.1 mg ml⁻¹ for 20 min at room temperature. The different samples were incubated at the indicated temperatures in SDS-PAGE sample buffer for 5 min prior to being subjected to SDS-PAGE. Proteins were analysed by Western blotting (WB) using anti-Flag antibody. (b) N–N interaction was examined in co-immunoprecipitation experiments. HEK 293T cells co-expressing N–HA and Flag–N, or each of the Flag-tagged N mutants, were lysed and cell extracts were immunoprecipitated using anti-Flag antibody (IP: α-Flag). Precipitated proteins were analysed by Western blotting, as indicated on the left of each panel. Expression levels of WT Flag–N, each N protein mutant or N–HA in whole-cell extracts (WCE) were examined by Western blotting, as indicated. Molecular masses of markers are indicated on the left in kDa.