Figure 3

Expression of Bucky ball after transfection of fusion plasmids in vitro and in embryonic stages around oviposition in vivo (a) Bucky ball fusion-constructs form insoluble fusion proteins. Bucky ball fusion construct and controls (EGFP/Venus only) were transfected in DF1 cells, respectively. After 5 days, the cells were harvested, and total extracts, insoluble, and soluble fractions were assessed by Western blotting with an anti-GFP antibody. Controls (EGFP/Venus only, lanes 1, 4, and 7), zebrafish Bucky ball-EGFP (lanes 2, 5, and 8), and chicken Bucky ball-Venus fusion constructs. M, size marker with visible bands at 20, 40, 50, 60, 100, 150 kDalton. Note, the Bucky ball fusion proteins appear in the insoluble fraction (lanes 4, and 5) and migrate much slower than expected for the calculated molecular weights of 101 kDa (zBucky ball-EGFP) and 116 kDa (cBucky ball-Venus). The EGFP/Venus control shows a protein at the expected molecular weight of 27 kDa. (b) Detection of embryonically transcribed Bucky ball in the chicken blastoderm. The upper panel refers to Bucky ball and the lower panel to GAPDH transcripts. Samples 1 to 3 were generated from stage EGK X²⁸ laid egg blastoderms, and samples 4 to 6 from 24 h incubated embryos (stage HH 6²⁷). DNA-ladder in lanes (M): NEB 100 bp ladder N3231, (B) negative control amplification without template.