FIG. 4.
In vivo binding of g5p to ToLCV DNA. (A) Flag epitope-tagged CP66:6G:g5 protein expressed in protoplasts was immunoprecipitated with anti-Flag antibody coupled to agarose after lysis of protoplasts in NP-40 buffer containing different concentrations of NaCl (shown above lanes 1 to 6) or RIPA buffer (lane 7), and the immunoprecipitated protein was detected on a Western blot with anti-Flag antibody (lanes 2 to 7). Lane 1 contained protein immunoprecipitated from protoplasts transfected with wild-type virus as a control. The protein band present in all lanes at ∼24 kDa is the light chain of anti-Flag antibody used for immunoprecipitations. The immunoprecipitated CP66:6G:g5 protein was detected at two different molecular masses corresponding to monomeric and dimeric forms. Positions of molecular mass markers are indicated in kilodaltons on the left. (B) Viral ssDNA that coimmunoprecipitated with the Flag epitope-tagged CP66:6G:g5 protein was detected on a Southern blot with 32P-labeled A-component DNA as a probe. Lanes 1 to 7 were given the same treatments as in panel A.