Figure 3. Association of HSC70 with polysomes is altered following heat shock.

(A) Western blot of HSC70/HSP70 (using pan-HSP70 antibody, see Experimental procedures for details) protein levels (middle panel) across a polysome gradient in Control and severe heat shock cells (HS2S, top panel). RPL10A was used to assess ribosome abundance in each fraction (middle panel). Levels of comigrating HSC70/HSP70 protein and RPL10A protein were measured using ImageJ software. Ribosomal association of HSC70/HSP70 was quantified by normalizing the amount of HSC70/HSP70 protein by the amount of RPL10A protein in each fraction (HSC70/HSP70 / RPL10A) for both control (blue bars) and heat shock (red bars) conditions. P:M ratio is 1.9 in Control and 0.18 in HS2S. Profile is representative of six experiments, where relative P:M ratios of HS2S were 8.5% ± 2.9% of Control. (B) Quantification and significance of the reduction in HSC70/HSP70 ribosome association following heat shock. Mean and standard error of the ratio (log₂) of HSC70/HSP70 association (HSC70/HSP70 signal/RPL10A signal), in HS2S to control conditions, in each fraction, over 5 biological replicate experiments. Fractions in which HSC70/HSP70 and/or RPL10A signal was undetectable were excluded from analysis (see Methods for details). Significance of reduction in HSC70 ribosomal association is given by t-test. * indicates p < 0.05; ** indicates p < 0.01.

(C) Hydrophobicity scores (see Supplemental Experimental procedures) of the first 70 amino acids encoded by all highly translated mRNAs (in black), of significantly paused mRNAs (in magenta) and of non-significantly paused mRNAs (in orange). The background representing the overall average Kyte-Doolittle hydrophobicity of all highly translated mRNAs (grey dashed line) is shown for reference. T-test p-value for the difference in the mean hydrophobicity values of the first 25 amino acids between all significantly paused mRNAs and non-significant mRNAs was 3.8 × 10⁻⁷. (D) Hsp70 binding site scores (Supplemental Experimental Procedures) were plotted as in (C). T-test p-value for the difference in the mean Hsp70 scores of the first 25 amino acids between all significantly paused mRNAs and non-significant mRNAs was 5.3 × 10⁻⁶.

See also Figure S3.