Fig. 6.

Solubility of APPL1. (A) Prolonged administration of MG132 increases insolubility of APPL1. HeLa cells were treated with DMSO or 10 μM MG132 for the indicated times. Cells were either lysed in hot sample buffer (for total cell lysates) or in RIPA buffer, in which case the detergent-soluble and -insoluble fractions were collected. Equal amounts of protein were resolved by SDS-PAGE and blotted for APPL1 (upper and lower panels depict long and short exposure times, respectively), EEA1, HDAC2 and α-tubulin. (B) HEK293 cells transfected with APPL1 and HA-ubiquitin (HA-Ub) were treated as indicated, lysed in RIPA buffer and extracted into the detergent-soluble and -insoluble fractions which were blotted for EEA1, APPL1 and HA. Immunoprecipitates of APPL1 and control rabbit immunoglobulins (Ig) from the soluble cell fraction were blotted for APPL1 and HA. Asterisks indicate a position of unmodified APPL1.