A) The effects of heparitinase and PMA can be reverted by adding 40 μM TAPI-0 to the incubation medium. MCF-7 cells were treated for 30 minutes and Triton-X100 soluble lysates containing 40 μg protein were subjected to immunoblot. The position of full length ErbB4 protein and ErbB4 80 kDa fragment as detected by the polyclonal sc-283 anti ErbB4 antibody are indicated by arrows. B) The effect of heparitinase is specific for TACE-cleavable (JM-a) ErbB4 isoform. MCF-7 cells were transiently transfected with ErbB4 JM-a CYT2HA or JM-b CYT2HA gene construct. Lysates were subjected to immunoblot with HA-specific monoclonal antibody. C) The effect of heparitinase treatment could be demonstrated in T47D cells treated similarly to MCF-7 cells. D) Degradation of chondroitin sulfate did not increase the formation of ErbB4 80 kDa fragment in T47D cells. E) Heat inactivated incubation medium from heparitinase treatment of T47D cells had only small effect on ErbB4 80 kDa fragment formation. The intensity of the ErbB4 80 kDa fragment staining as indicated in C and D was quantified with ImageJ software vs. 1.38 (NIH, USA). Beta-actin was used as load control (not shown). Abbreviations: Htase, heparitinase; Ctase, chondroitinase, PMA, phorbol myristyl acetate. Ht med. heat inactivated heparitinase incubation medium. The images are representative of at least three independent analyses.