Fig. 2. Protein blots of extracts from KNOLLE transgenic plants. Protein extracts were separated by SDS-PAGE and analysed by immunoblotting with anti-KN antiserum (see Materials and Methods). (A) Misexpression of KNOLLE from the flower-specific AP3 promoter. Lanes 1-4: total protein extracts of wild-type (wt) flowers (lane 1) and AP3::KN1 transgenic petals (lanes 2-4). Lanes 5-7: cell fractionation of pooled extracts (lanes 2-4). (B) Organ distribution of KNOLLE protein misexpressed from the CaMV 35S promoter. 35S, T3 transgenic plants homozygous for 35S::KN; wt, wild-type control. (C) Quantitative analysis of KNOLLE expression in 20 days old seedlings grown on different media. Lanes 1-3: 0.5x MS salts with 0%, 1%, 3% sucrose. Lanes 4-6: 1x MS salts with 0%, 1%, 3% sucrose. Upper panel: similar amounts of total protein were loaded. Lower panel: 35S::KN samples 1 and 3 were diluted 1:100 and 1:500; wild-type (wt) samples 2, 3 and 5 were not diluted. (D) Membrane integration properties of KNOLLE protein from T3 generation plants homozygous for the 35S::KN transgene. Protein extracts from leaves of 35S::KN plants (35S, top) or from flowers and siliques of wild-type control (wt, middle) were differentially centrifuged. The P100 fraction (lane 3) was resuspended with or without Triton X-100 followed by a second 100,000 g centrifugation (lanes 4-7). Plants heterozygous for the knolle mutant allele UU1319 (bottom) express normal KN protein (arrow) and a truncated KN protein without the membrane anchor that was not pelleted by 100,000 g centrifugation (arrowhead). Arrow: position of the 34 kDa KN protein; S10, supernatant of 10,000 g centrifugation; S100, supernatant; P100, pellet of 100,000 g centrifugation.