FIGURE 6.

The processing and trafficking of PAM-1/H3A is less sensitive to dissipation of intracellular pH gradients than PAM-1. A, cells were treated with alkalinizing agents or concanamycin A for 24 h and lysates were collected into TMT when spent medium was harvested. For cell lysates, equal amounts of protein (5 μg) from control (C), dimethyl sulfoxide (DMSO) vehicle (D), 2.5 mm ammonium chloride and 5 mm methylamine (N) and 1 nm concanamycin A (CA)-treated cells was fractionated by SDS-PAGE; an aliquot (1%) of the spent medium was also analyzed. PAM was visualized using an antibody specific to PHM (*, nonspecific band). Processing was evaluated by plotting the sPAM/PAM-1 (B) and sPHM/PAM-1 (C) ratios from duplicate samples from two independent experiments; error bars show range. D, the amount of sPHM collected in each 24-h medium sample was compared with the amount of sPHM in the cell lysate; data are expressed as % sPHM secreted/h. #, treatment effect significant for PAM-1, p < 0.05; *, PAM-1/H3A different from PAM-1 for that treatment group, p < 0.05.