Fig 2: Co-chaperones bind to aggregation-prone site 2 but not to site 3.Site 2 (a) and site 3 (b) peptides indicated by TANGO were inserted into the ER-C?-QVT construct and co-expressed with either GRP170, ERdj5, or ERdj4-3HA. Peptide-containing constructs were isolated with anti-?, separated by reducing SDS-PAGE, and transferred for blotting with the indicated antibodies. A construct with no peptide insert (? ins) was used as a negative control, and a construct (mHC#2) from original study was used as a positive control. Sequence of mutants and TANGO plots are shown in Supplementary Fig. 3. Since site 3 included L188, which is mutated in ILD, constructs were also made with glutamine (Q188) at this position. Source data are provided as a Source Data file. Representative images for n = 3.

Fig 3: Mutation of site 2 in the L188Q mutant reduces co-chaperone binding, but significant binding remains.a Cells were co-transfected with cDNAs encoding L188Q (parental), the indicated L188Q mutants in which site 2 and site 3 mutations had been engineered, along with either GRP170, ERdj5, or ERdj4-3HA. Twenty-four hours after transfection, cells were harvested and lysed with the appropriate buffers as described in the methods section. 1.5% of the lysates were retained for input, and the remaining cell lysates were immunoprecipitated with anti-SP-C. Samples were separated by reducing SDS-PAGE, and both SP-C and associated co-chaperones were detected by western blotting with specific antibodies. b Signals for binding of each co-chaperone to the site 2 mutant were first normalized to mutant L188Q expression levels compared to that of parental L188Q. The binding of each co-chaperone to the mutant constructs (circle) was expressed as a percent of that bound to the parental construct (triangle). For GRP170 binding, n = 3 independent experiments, for ERdj5, n = 4 independent experiments, and for ERdj4, n = 5 independent experiments. Standard deviation (SD) and standard error of the mean (SEM) were determined. Horizontal lines and error bars represent the mean and (SEM), respectively. The difference in co-chaperone binding between SP-C L188Q (parental) and each SP-C mutant (SP-C L188Q site 2, site 3, and site 2/3) was assessed using one-sample t test. Differences between the binding of co-chaperones (GRP170 vs ERdj5, ERdj5 vs ERdj4, and GRP170 vs ERdj4) to each SP-C mutant were estimated using unpaired t test. A threshold p < 0.05 was statistically significant. Source data are provided as a Source Data file.