Figure 4
From: Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers
SARS-CoV N protein is also forming oligomers. (a) Bacterial extract from E. coli expressing 6xHis-tagged SARS-CoV N protein (input) was incubated with immobilized GST or GST-SARS-CoV-N protein. Precipitated proteins were eluted in SDS-sample buffer and analyzed by western blot using an anti-His monoclonal antibody. The amounts of GST and GST-N were assessed by staining the PVDF membrane with Ponceau Red. A260/280 ratios <0.1 indicated the absence of nucleic acids in the samples. Only part of the western blot images is shown in the figure. (b) Bacterial extracts from E. coli expressing 6xHis-tagged SARS-CoV N1-N2a were processed and analyzed as in panel (a). A260/280 ratios <0.1 indicated absence of nucleic acids in the samples. Only part of the western blot images is shown in the figure. (c) Bacterial extracts of E. coli expressing 6xHis-tagged SARS N protein and N1-N2a truncation were sedimented in a 5–20% glycerol gradient at 50,000 g for 75 min. Eleven fractions were collected and protein content analyzed using antibodies against the 6xHis tag. Gradient and centrifugation conditions were assessed by sedimenting a LR7 cell extract and probing the collected fractions with a GAPDH antibody. Only part of the western blot images is shown in the figure. (d) Quantification of the immunoblots presented in panel (c) plus standard deviation (SD) (n = 3).
