Fig 1: HAs with tetrabasic and wild-type HPAIV MBCS are cleaved by duck furin. (A) Alignment of cleavage site mutants in the H5 HA from HPAIV A/Indonesia/5/05. HA1 and HA2 are separated by an asterisk. (B) Furin-deficient LoVo cells were transfected with empty vector (EV) or plasmids coding for human (HF), chicken (CF), or duck furin (DF). Cells were harvested and lysed for western blot analysis targeting furin at 24 h posttransfection. Proteins were deglycosylated and size-separated on a 10% reducing and denaturing SDS-PAGE gel. Furin was detected using a rabbit monoclonal antibody. Beta-actin was included as loading control. Protein size in kilodaltons was assessed by running the samples alongside a ladder. Representative blots of three independent experiments are depicted. (C) LoVo cells were cotransfected with plasmids coding for HF, CF, or DF and either HA-WT (WT) or HA-ΔMBCS (Δ). Cells were treated with TPCK-trypsin (2.5 μg/mL) for 10 min as positive control and EV was transfected as negative control. Cells were harvested and lysed for western blot analysis at 48 h posttransfection and size-separated on a 10% reducing and denaturing SDS-PAGE gel. HA0 and HA1 were detected using a rabbit polyclonal H5N1 serum. Beta-actin and a protein ladder were included as for panel B. Representative blots of three independent experiments are depicted. (D) Lovo cells were cotransfected with plasmid coding for human, chicken, or duck furin and HA containing intermediate cleavage site motifs REKR, RKTR, and RKKR, and processed as described for panel C. (E) Quantification of HA cleavage percentages. Bars represent the means inferred from the quantification of western blots from three independent experiments. Error bars represent the standard deviations (SD).