Figure 1. Proteotoxic stress attenuates protein synthesis by affecting translation elongation.

(A)Global protein synthesis in HEK293 cells treated with either 10 mM AZC, or 20 μM MG132, or both. [³⁵S] radioactivity of trichloroacetic acid (TCA)-insoluble material was measured at given times. Means ± SEM of four experiments are shown.

(B) Polysome profiles were determined using sucrose gradient sedimentation. HEK293 cells were pre-treated with either 10 mM AZC, or 20 μM MG132, or both for 60 min followed by polysome preparation. P/M ratio is calculated by comparing areas under the polysome and 80S peak.

(C) HEK293 cells were treated with increasing doses of AZC (from 0 to 25 mM with 5-fold dilution) in the presence of 20 μM MG132 for 60 min, or increasing doses of NaAsO₂ (from 0 to 1 mM with 2-fold dilution) for 60 min (left two panels), followed by immunoblotting using antibodies as indicated. The right two panels show the immunoblotting results of cells treated with 10 mM AZC and 20 μM MG132 or 500 μM NaAsO₂ for various times (0, 10, 30, 60, 120, and 180 min)

(D) The ribosomal half-transit time was determined in the absence or presence of 10 mM AZC and 20 μM MG132. Fitting lines of [³⁵S] incorporation into total (filled circle) and completed (open triangle) protein synthesis are obtained by linear regression. Means ± SEM of three experiments are shown.

(E) Schematic for nascent chain immunoprecipitation assay to differentiate elongation defect from initiation deficiency (left panel). HEK293 cells expressing Flag-GFP were pre-treated with 10 mM AZC and 20 μM MG132 or 500 μM NaAsO₂ for various times (0, 10, 30, 60, 120, and 180 min). Immuno-precipitation was performed using anti-Flag antibody-coated beads followed by immunoblotting with anti-RpS6 antibody. The 0 time point serves as the control condition without any drug treatment. See also Figure S1.