Figure 2
Figure 2. Reduced proteasome activity and increased workload in PI-sensitive MMCs. (A) Proteasome activity in the relatively sensitive KMS.18 and MM.1S and the relatively resistant U266 and RPMI8226 lines. Proteasome-specific chymotryptic, trypsin-like, and caspase-like activities were assessed in cell extracts and expressed on a per-protein basis. The histogram shows the relative quantification of all 3 activities within each line, and levels relative to the corresponding activity in U266 (*P < .001). The average of at least 4 independent experiments (± SD) is shown. (B) Proteasome β-catalytic and α subunits in MM lines. Extracts from U266, RPMI8226, KMS.18, and MM.1S cells were resolved by SDS-PAGE and blotted with Abs to different proteasome catalytic β and noncatalytic α subunits (1 of 3 representative experiments). Equal protein amounts were loaded in each lane, with a background stable band serving as a loading control (bckgd). The right panel shows the relative densitometric quantification of catalytic β subunits in 3 independent experiments (average ± SD), on correction by the band intensity of β-actin in the corresponding blot. (C) Immunofluorescence against LMP7 in U266 and MM.1S cells reveals higher immunoproteasome levels in the relatively less sensitive U266 cells. (D) Higher proteasome workload and protein synthesis in PI-sensitive MMC. U266, RPMI8226, KMS.18, and MM.1S cells were pulsed for 5 minutes with 35S amino acids and chased for 30 minutes, with or without PIs (lactacystin, bortezomib, and epoxomicin; 2 μM each). Proteasome-mediated degradation of newly synthesized proteins was calculated as the percentage of TCA-insoluble radioactivity, the disappearance of which during the chase was inhibited by PIs, relative to the total radioactivity present at the end of the pulse, as in Figure 1. The left panel shows proteasomal degradation in all MM lines, while the right panel quantifies the proteosynthetic activity of each line as the incorporation of hot amino acids into TCA-insoluble polypeptides at the end of the pulse. An average of 3 independent experiments (± SD) is shown. *P < .001.

Reduced proteasome activity and increased workload in PI-sensitive MMCs. (A) Proteasome activity in the relatively sensitive KMS.18 and MM.1S and the relatively resistant U266 and RPMI8226 lines. Proteasome-specific chymotryptic, trypsin-like, and caspase-like activities were assessed in cell extracts and expressed on a per-protein basis. The histogram shows the relative quantification of all 3 activities within each line, and levels relative to the corresponding activity in U266 (*P < .001). The average of at least 4 independent experiments (± SD) is shown. (B) Proteasome β-catalytic and α subunits in MM lines. Extracts from U266, RPMI8226, KMS.18, and MM.1S cells were resolved by SDS-PAGE and blotted with Abs to different proteasome catalytic β and noncatalytic α subunits (1 of 3 representative experiments). Equal protein amounts were loaded in each lane, with a background stable band serving as a loading control (bckgd). The right panel shows the relative densitometric quantification of catalytic β subunits in 3 independent experiments (average ± SD), on correction by the band intensity of β-actin in the corresponding blot. (C) Immunofluorescence against LMP7 in U266 and MM.1S cells reveals higher immunoproteasome levels in the relatively less sensitive U266 cells. (D) Higher proteasome workload and protein synthesis in PI-sensitive MMC. U266, RPMI8226, KMS.18, and MM.1S cells were pulsed for 5 minutes with 35S amino acids and chased for 30 minutes, with or without PIs (lactacystin, bortezomib, and epoxomicin; 2 μM each). Proteasome-mediated degradation of newly synthesized proteins was calculated as the percentage of TCA-insoluble radioactivity, the disappearance of which during the chase was inhibited by PIs, relative to the total radioactivity present at the end of the pulse, as in Figure 1. The left panel shows proteasomal degradation in all MM lines, while the right panel quantifies the proteosynthetic activity of each line as the incorporation of hot amino acids into TCA-insoluble polypeptides at the end of the pulse. An average of 3 independent experiments (± SD) is shown. *P < .001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal