Figure 3.

Effects of hypertonic stress on ubiquitination of AQP1. (A) Fibroblasts were incubated in isotonic or hypertonic medium for 8 h, and protein immunoblots of total cell lysates were probed with anti-ubiquitin antibodies. (B) Fibroblasts were incubated in isotonic (control) or hypertonic medium (+200S) for 8 h, and immunoprecipitation with protein A-Sepharose was performed by using rabbit (IgG) or anti-AQP1 antibodies (anti-AQP1). Protein immunoblots of the immunoprecipitates were probed with anti-ubiquitin antibodies. Arrowheads mark the position of discrete bands of ubiquitinated AQP1. (C) Cells were incubated in isotonic (control) or hypertonic medium (+200S) for the designated times, AQP1 was immunoprecipitated as described, and immunoblots were probed with anti-AQP1 (Upper) or anti-ubiquitin antibodies (Lower). Arrowheads mark discrete bands labeled by anti-ubiquitin antibodies.