Figure 4.

CrPV-1A interacts with Argonaute 2 in S2 cells. (a) S2 cells were transfected with plasmids transiently expressing CrPV-1A-3FH (lane 2, 4) or empty vector control (lane 1, 3). Flag-His tagged CrPV-1A was immunopurified with anti-Flag antibody and western blotted with Drosophila Argonaute 2 (α-Ago2, right panel) or Argonaute 1 (α-Ago1, left panel) polyclonal antibodies. Two isoforms of Ago2 (Ago2a and Ago2b) were detected with Ago2 specific antibodies. Asterisks (*) indicate cross-reacting host-protein band. Lane 6 and 8 show the positive detection of E.coli expressed Drosophila Ago1 and endogenous Drosophila Ago2 with Ago1 and Ago2 antibody respectively (Ab control). (b) Flag-His tagged CrPV-1A148, CrPV-1A108 were transfected in to S2 cells, immunopurified using anti-Flag antibody and probed with Ago2 antibody (α-Ago2). To detect the expression of 1A protein, the blot was stripped and further probed with anti-Flag antibody (α-Flag) (lane 3 and lane 4, bottom panel). Asterisks (*) indicates a cross-reacting host-protein band (c) Stable S2 cell lines either expressing CrPV-1A-3FH (induced with CuSO₄) or uninduced control was lysed, immunopurified with anti-Flag antibody followed by Talon magnetic bead purification. The silver stained gel shows the co-purification of the CrPV-1A and Argonaute 2 (Ago2b isoform) from S2 cells (right lane). Left lane represents the molecular weight marker. Asterisk (*) indicates non-specific contaminant band (see Supplementary figure 3).