Fig 2: Thermotolerance depends on both proteasome and HSP70 activities Similar experiment as described in (Fig 4A) with naïve MCF7 and thermotolerant MCF7aTT cells as indicated. Means ± SEM, n = 3, *P < 0.05, **P < 0.01 (calculated with the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli).Same experiment as in (A) with thermotolerant MCF7aTT cells incubated with the HSP70 inhibitor VER-155008 (50 µM, 1 h) or MG132 (20 µM, 1 h). The signal obtained for ribosomal protein RPS19 and RPL7 is shown for reference, documenting residual polysomes despite the flat UV trace. Right panels are quantifications of K48-ub (means ± SEM, n = 2), black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity. The bar graphs represent the P/M ratios (means ± SD, n = 3, numbers above bars indicate P values calculated with the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli).Sucrose density gradient fractions described in (B) were assayed for partitioning of HSPA1 and HSPH1 by immunoblotting. HSPA1 data were quantified in a line graph (means ± SEM, n = 4).Cell lysate was spun through a 30% sucrose cushion to pellet polysomes. Nascent proteins were released from polysomes by dissociating 80S ribosomes with EDTA (see Appendix Fig S3). Soluble and insoluble fractions were prepared by centrifugation and assayed for the level of K48-linked polyubiquitin. The levels of K48-linked polyubiquitin were quantified (means ± SD, n = 3). Immunoblots within black squares were run on the same gel but irrelevant lanes were removed as indicated by white lines. Source data are available online for this figure.

Fig 3: Acquisition of the STING TRAF6 Module Remodels Downstream Immune Responses(A) Schematic representing generation of mouse macrophage cell lines with different STING gene alleles.(B) Western blot analysis of STING expression in different mouse macrophage cell lines compared with the RPS19 loading control. Data are representative of three biological experiments.(C) Heatmap of reads per kilobase of transcript per million mapped reads (RPKM) of 100 differentially upregulated genes in mouse macrophage cell lines: control, mouse STING (mSTING) ?CTT, mSTING, and mSTING zebrafish C-terminal tail (zfCTT). Acquisition of the zebrafish STING CTT enhances the STING-dependent interferon response and additionally activates alternative NF-?B responses.(D) Venn diagram depicting upregulated genes, the normal mouse STING response, and the alternative mSTING zfCTT response.(E) Visualization of RNA sequencing (RNA-seq) gene coverage and transcript levels for the selected differentially expressed genes Ifnb, Il12b, nos2, and ccl12 in macrophage cell lines: control, mSTING ?CTT, mSTING, and mSTING zfCTT using an integrative genomics viewer (IGV) (Robinson et al., 2011).(F) Transcript-level analysis of selected genes by qRT-PCR. qRT-PCR data are representative of three independent experiments.Data are plotted with error bars representing the SD of the mean.

Fig 4: HSP70 promotes proteasome recruitment to polysomes Trypsin-like proteasome activity determined in sucrose density gradient fractions as described in Fig 4A using an in vitro assay. Results are shown relative to the proteasome activity obtained in MCF7 cells maintained at 37°C (means ± SEM, n = 4).MCF7 cells were exposed to the proteasome inhibitor MG132 (20 µM) or 42°C for 1 h after which an equal amount of cell lysate was spun through a 30% sucrose cushion. Resulting polysomes were assayed for the levels of 26S proteasome subunits and HSPA1.Cell lysates from MCF7 cells maintained under the conditions indicated were fractionated by sucrose density gradient centrifugation. Polysomal fractions were subjected to immunoprecipitation with RPS19 antibody, and precipitates were analyzed by quantitative LC-MS/MS. The heat map represents normalize spectral abundance factors (NSAF).Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells, and puromycin labeled peptides were pulled down as illustrated in the scheme. Precipitates were assayed for the presence of K48-linked polyubiquitin, chaperones, and proteasome subunits by immunoblotting.Partitioning of the 20S proteasome subunit PSMB1 along the sucrose gradient under the indicated conditions was assessed by immunoblotting, black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity. The line graph on the top shows trypsin-like proteasome activity in the same fractions (means ± SD, n = 3). Source data are available online for this figure.

Fig 5: Systematic identification of protein-binding RNA elements from large-scale RNA structural architecture.a Schematic of RNA immunoprecipitation with an anti-EIF3B antibody and RNA structure library, version 2. The numbers of target RNA structures are described in parentheses. The 293FT cell-derived EIF3 complexes were immobilized on the antibody-conjugated beads. b RNP interactions of the EIF3 complex. The data were calculated from two independent experiments. A histogram of binding intensities with the positive control (green line) and Z-score = 2.0 (blue line) is shown. The terminal motif derived from HIV-1 gag IRES was a positive control. The colored bar indicates the binding intensities for the functional annotation. c A structure-wide functional annotation of the HIV-1 RNA secondary structures. The color of each structure was determined by the binding intensity; the library does not include RNA structures that contain gray-colored RNA residues. d Representative EIF3-complex binding structures from human 5'UTRs. e Schematic of the pull-down assay with an RNA–DNA–biotin hybrid. The target RNA structure was immobilized on a streptavidin-coated magnetic bead using a biotinylated tethering DNA strand. Enriched proteins in the bound fraction were collected by magnetic separation and analyzed by capillary-based immunodetection. f Gel images of the microcapillary-based immunodetection with anti-EIF3B, anti-RPS19, and anti-ACTB antibodies. The image shows representative data from two independent experiments. Average molecular weights were calculated from size markers and ladders. Source data are provided as a Source Data file.