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. 2022 Dec 1;132(23):e156967. doi: 10.1172/JCI156967

Figure 7. Correlation of predicted protein instability with ECM deposition.

Figure 7

(A) The propensity for THBS1R1034C aggregation in the ECM was further assessed through ectopic expression of FLAG-tagged THBS1 in COS-7 cells. The absence of DAPI (nuclear acid) and F-actin (cytoskeleton) staining indicated the successful removal of cellular components. THBS1 extracellular deposition was evaluated by anti-FLAG IF staining. Consistent with the in vivo findings, there was greater accumulation of mutant THBS1C1034 in ECM than of THBS1R1034 (WT). Scale bar: 10 μm; enlarged insets, ×4. (B) Plasmids with Arg1034 mutated to Lys, Gln, Ser, Ala, and Gly were transfected into COS-7 cells. ECM deposition was lowest in Lys1034 and greatest in Gly1034, with Gln, Ser, and Ala showing intermediate effects. Scale bar: 10 μm. (C) THBS1 ECM deposition strongly correlated with the change in protein stability (R2 = 0.86, P < 0.0001, by linear regression), suggesting that Arg1034 had an essential role in reducing abnormal THBS1 ECM deposition. The change in protein stability (ddGB) was derived from 5 analyses in FoldX. Protein deposition was quantified on the basis of fluorescence intensity of anti-FLAG immunostaining from 3 independent experiments. The mean values of ddGB and fluorescence intensity were used for regression analysis. Error bar indicates the SEM.