Figure 6.

S₄-SLFL2 can ubiquitinate self and nonself S-RNase. A, An S₄-SLFL2 antisense oligonucleotide gene was transfected into cv 21-21 and cv 21-26 pollen tubes for ubiquitin assays with self S-RNase. Error bars represent sd calculated from two biological replicates (separate biological material). Asterisks indicate significance by Student’s t test (**P < 0.01). B, The self S-RNase purified from cv 21-21 pistils was incubated with cv 21-21 and cv 21-26 pollen tube proteins and MG132 was added. The protein samples were stained by Coomassie Brilliant Blue (CBB), and S₄-SLFL2 was detected by S₄-SLFL2 antibody. C, An antisense S₄-SLFL2 oligonucleotide gene was transfected into cv 21-21 and cv 21-26 pollen tubes for ubiquitin assays with nonself S-RNase. Error bars represent sd calculated from two biological replicates (separate biological material). Asterisks indicate significance by Student’s t test (**P < 0.01). D, The nonself S-RNase purified from cv Hongdeng (HD) pistils was incubated with cv 21-21 and cv 21-26 pollen tube proteins and MG132 was added. The protein samples were stained by Coomassie Brilliant Blue, and S₄-SLFL2 was detected by S₄-SLFL2 antibody. α-S₄-RNase, S₄-RNase antibody; α-S₄-SLFL2, S₄-SLFL2 antibody; α-ubiquitin, ubiquitin antibody.