Tensin3 H1 binds directly to the R11 domain of talin. (A and B) Immunostaining of (A) endogenous talin in vinculinKO mouse embryonic fibroblasts (vin−/− MEFs) or (B) endogenous vinculin in talin1/2 KO cells expressing mCh-tensin3-FL-cBAK reveals that vinculin interacts with tensin3 indirectly through talin. (C) Schematic of talin constructs used. (D) Representative images of vin−/− MEFs cells expressing GFP-talin∆R1-R10 or GFP-talin∆FERM together with mCh-tensin3-FL-cBAK. (E) Representative images of talin1/2 KO cells expressing GFP-talin-Cterm with either mCh-tensin3-FL-cBAK or mCh-tensin3-FL∆H1-cBAK. (F) Sequence alignment of the talin binding sites in DLC1 and RIAM with the H1 sequence of TNS3. (G)¹H,¹⁵N-HSQC NMR spectra of 400 µM ¹⁵N-labelled isolated talin R11 in the absence (blue) and presence (red) of 1,600 µM of tensin H1 peptide. Note the decrease of signals in the complex due to the exchange broadening. (H) ITC measurements of tensin3 H1 interactions with talin R11. The experiments were conducted with 80 µM of R11 in cell and 1.2 mM of the peptide in the syringe. Scale bars in A, B, D, and E indicate 5 µm.