Analysis on individual transgenic plants harboring single copy of transgene. (A) Expression analysis of hG-CSF on individual transgenic plant leaves harboring single copy of transgene by real time PCR (upper panel) and immunoblot analysis (middle panel). Lower panel, the expression levels in individual plants determined by immunoblot as shown in middle panel. The triangle symbol marked the location of the molecular weight at 18.6 kD in accordance with comercial hG-CSF standard. All values in upper panel were normalized with 18S rRNA level and relative to the lowest expression level in SH transgenic plants. Grey squares represented the average expression of the construct based on 3 individual plants (1, 2, 3) and P values were obtained by two-tailed Student t-test on expression levels in the population of Constructs SH and USH. (B) Immunoblot analysis of endogenous ubiquitin in non-transgenic (WT) and trangenic plants using antibody against ubiquitin. Upper panel, dot immunoblot analysis of total soluble protein (2 μg/dot) in tobacco leaves, and lower panel, immunoblot analysis of total soluble protein (10 μg/lane). Free ubiquitin, a 8.5 kD protein, was indicated by arrow, and ubiquitin-cojugated proteins, including the smear high molecular weight component, were denoted by the bracket. Lanes SH and USH, see Figure 1.