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. Author manuscript; available in PMC: 2014 Feb 7.
Published in final edited form as: Mol Cell. 2013 Jan 3;49(3):453–463. doi: 10.1016/j.molcel.2012.12.001

Figure 3. Disrupting endogenous Hsc70 recapitulates the effects of proteotoxic stress on early elongation pausing.

Figure 3

(A) Sucrose cushion analysis of ribosome-associated Hsc70 along with AZC and MG132 treatment. Both the total and ribosome pellet were immunoblotted using antibodies as indicated.

(B) Global protein synthesis was analyzed in HeLa-tTA cells infected with adenoviruses expressing Hsc70(WT) and Hsc70(K71M). Transgene expression was induced by 12 h Dox removal. [35S] radioactivity of TCA-insoluble material was measured at given times. Means ± SEM of three experiments are shown.

(C) Polysome profiles were determined from cells as in (B) using sucrose gradient sedimentation.

(D) Meta-gene analysis for early elongation pausing in cells with or without Hsc70(K71M) expression. Normalized RPF reads are averaged across the entire transcriptome, aligned at either their start (left panel) or stop (right panel) codon.

(E) Both the 5′ and 3′ PI of each transcript in cells with or without Hsc70(K71M) expression are shown in box plots.

(F) Changes of 5′PI and 3′PI after Hsc70(K71M) expression. The log2 change is computed across the entire transcriptome and presented as a scatter plot with green dots for genes encoding ribosome subunits and red dots for mitochondria- encoded genes. See also Figure S5.