Vp 5s sonicator

Halothece 7418 cells were collected from 50 mL of the culture during the log phase (OD₇₃₀ = 0.6~0.9) and then stored at −80 °C until use. Cells were suspended in 700 μL of 50 mM Tris-HCl (pH 8.0) and sonicated on ice by using a VP-5s sonicator (TAITEC, Saitama, Japan) for a total of 40 s (repeated time-on/time-off of 10 s each time), with the output power set to 7. The samples were then centrifuged at 22,000 × g for 10 min at 4 °C. The supernatant solutions were used as total soluble protein extracts. Protein concentration was determined by using a TaKaRa Bradford Protein Assay Kit (Takara Bio Inc., Shiga, Japan).

The recombinant protein of human pacsin 2 SH3 domain (amino acids 426–486) used as bait for the GST pull-down assay was expressed and purified with the GST Gene Fusion System (Cytiva) as a GST fusion using Glutathione Sepharose 4B (17075601, Cytiva) according to the manufacturer's instructions. The GFP-tagged cytoplasmic domain of N-cadherin (amino acids 746–906) was expressed in HEK293T cells, and the cell extract was prepared by sonication using a TAITEC VP-5S sonicator (output: 5; 3×5 s) in extraction buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 2% Triton X-100, 1 mM PMSF and protease inhibitor (11697498001, Roche)] followed by centrifugation at 20,600 g for 10 min at 4°C. For the pull-down assay, 170 µl of the cell extract was added to 10 µl (bed volume) of GST–pacsin 2 SH3 or GST beads in the extraction buffer and mixed for 1 h at 4°C with gentle agitation. After washing three times with the extraction buffer, the bound proteins were analysed by immunoblot analyses.