The human Rsu1 was expressed in insect cell expression system. The DNA sequence of Rsu1 or its mutants was amplified based on PCR and inserted into pFastBac HTB vectors. The constructs were transfected into DH10Bac competent cells for preparing Baculovirus genome (bacmid). The recombinant bacmid was then transfected into Sf9 for generating P1 virus and further amplified after two passages. The high tilter P3 virus was used to infect Sf9 cells for 72 hr. The infected cells were harvested and lysed by high-pressure homogenizers in the buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 5 mM imidazole, 1 mM PMSF freshly supplemented). The supernatant isolated from lysates by centrifuging at 20,000 rpm for 30 min was loaded to Ni2+-NTA column. The eluted proteins were further purified by size-exclusion chromatography (GE Healthcare) with the buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 2 mM DTT). The DNA sequence of human PINCH1_LIM45C (aa.188–325), LIM5C (aa.249–325) or mutants, and human PINCH2_LIM5C (aa.276–363) were amplified by PCR methods and inserted into pET32a vector with an N-terminal thioredoxin (Trx)-His for expression in BL21(DE3) E. coli cells. The purification procedure was essentially the same as used for Rsu1.

For preparing the complex samples, Rsu1 and PINCH1 protein were mixed at 1:1.5 molar ratio, incubation with TEV and PreScission protease to cut the tags, and further isolated by Superdex75 size exclusion column (GE Healthcare). For the expression of IPP complex, the pETDuet vector contained His-SUMO-tagged human ILK (residues 1–452) and His-tagged human α-parvin (residues 1–372) was co-transformed with the pRSF-SUMO vector inserted with human PINCH1(residues 1–325) in Rosetta (DE3). The cells were expressed in 16°C for ~18 hr and then were harvested for the purification. The complex protein was purified by Ni2+-NTA column, followed by adding sumo protease to the elution to remove the SUMO-tag. The protein sample was further purified by size-exclusion chromatography (GE Healthcare) with the buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 2 mM DTT). The IPP complex fractions after size-exclusion chromatography was verified by SDS-Page gel and collected. The proteins were refrigerated in −80°C for the following applications.

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