FIGURE 1.

Proteasome-P97 interaction. A, left panel shows a time course induction of P97-proteasome interaction upon arsenite treatment (0.5 mm). Untreated or arsenite-treated lysates were subjected to PSMA1 (26 S) IP followed by a PSMD14 and P97 immunoblots. Cycloheximide (10 μg/ml) was added to prevent de novo protein synthesis. Right panel indicates the protein content (as evaluated by Coomassie-stained SDS-PAGE) of the 26 S proteasomes purified from control and arsenite-treated cells. Input represents 50 μg of total cell extract while the IP was performed using 2 mg of cell extract. B, HEK 293 cells were treated with several stress agents and subjected to PSMA1 (26 S) IP and P97 immunoblot. The stress agents indicated are: 2, the electrophile methylmethanesulfonate (MMS, 2.4 mm 1 h); 3, the amino acid analog Azetidine (5 mm, overnight); 4, proteasome inhibitor MG132 (10 μm, 1 h); 5, the ER stress-inducing agents tunicamycin (Tm, 20 μg/ml, 1 h); 6, thapsigargin (Tg, 2 μm, 1 h) 7, arsenite (0.5 mm, 1 h); and 8, MG132 (10 μm, 1 h)+arsenite (0.5 mm, 1 h). Lane 1 represents the untreated cells. As a negative control we used a non-relevant IP against cells treated with arsenite. Right panel shows the quantitation of the P97 signal indicating the accumulative effect of MG132 and arsenite. Input represents 50 μg of total cell extract while the IP was performed using 2 mg of cell extract. C, immunoblot of P97, the 19S lid component PSMD14, the 20 S subunit PSMA1 and GAPDH in fractions of glycerol gradients prepared from untreated and Velcade-treated cells (1 h 10 μm). The immunoblot of P97 in PSMA1 immunoprecipitations from fractions of glycerol gradients is shown in the bottom panel. The migration of complexes of known size is indicated above. The migration of P97 from the indicated fractions was quantified and presented as fractions from the total signal. Note the increase in P97 in the HMW fractions upon Velcade treatment. GAPDH IB served as a nonspecific migration control.