Fig. 6. Examination of proHP6 proteolytic processing by Factor Xa-treated proHP1b_Xa·.

(A) Proteolysis of proHP6 by Factor Xa at different levels. As controls, aliquots of proHP6 (1 μl, 100 ng/μl) were incubated with 0 to 400 ng Factor Xa at 37°C for 90 min. After 12% SDS-PAGE, immunoblot analysis was performed using 1:1000 diluted HP6 antisera as the primary antibody. (B) Cleavage of proHP1b_Xa by Factor Xa. One μl of 150 ng/μl proHP1b_Xa was incubated with 1 μl Factor Xa at 0 to 400 ng at 37°C for 90 min, treated with SDS-sample buffer with (left panel) or without (right panel) DTT, and separated by 12% SDS-PAGE gel followed by immunoblotting using HP1 antibodies. The predicted 30 kDa catalytic and 16 kDa regulatory domains are indicated with arrows. (C) Processing of proHP6 by proHP1b_Xa mutant and Factor Xa. The three proteins (50 ng each) were incubated at 37°C for 90 min and separated by reducing SDS-PAGE. Diluted HP6 antiserum was used for immunoblot analysis. Positions and sizes of the M_r markers are indicated on the left.