Flag peptide

Escherichia coli BL21(pLysS) cells harboring the pGEX-4T-1-VP3 plasmid were cultured separately in 200 ml of Luria-Bertani (LB) medium and induced with 1 mM isopropyl β-d-thiogalactopyranoside (IPTG; Sangon Biotech, Shanghai, China) at 16°C, with shaking at 90 rpm, overnight. Cell pellets were lysed by sonication in the binding buffer (50 mM Tris-Cl, 150 mM NaCl, pH 8.0). After centrifugation at 12, 000 × g for 10 min, the supernatant was incubated with GST resin for 4 h at 4°C, and then the beads were washed with ice-cold binding buffer. Finally, the protein was eluted in binding buffer containing 2 mg/ml reduced glutathione (A100399-0005; Sangon Biotech). For Flag-TRAF3 protein, HEK293T cells were transfected with Flag-TRAF3 for 24 h. Cells were lysed with NP-40 lysis buffer (P0013F; Beyotime, China) containing phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 12,000 × g for 10 min, the supernatant was incubated with anti-FLAG M2 affinity gel (A2220; Sigma-Aldrich) for 4 h at 4°C. The beads were washed five times with ice-cold phosphate-buffered saline (PBS) at 4°C. Finally, the protein was eluted with 0.5 mg/ml 3*FLAG peptide (A6001; APExBIO, Houston, TX, USA).

293T cells were transfected with Flag-tagged plasmid constructs and after 36 h harvested with lysis buffer followed by sonication on ice. After centrifugation at 13,800 × g for 20 min, the supernatant was filtered using 0.2 μm syringe filter and then incubated with anti-Flag M2 (Sigma, California, USA) by gently rotating overnight at 4 °C. The beads were then washed twice with Lysis buffer and twice with TBS and incubated with 3 × Flag-Peptide (APExBIO, Huston, USA) by gently rotating for 1 h at 4 °C to elute the bound proteins. The elution was done twice to maximize the recovery. The protein thus purified was condensed.

293FT cells were transfected with CADM4-FLAG, CADM1-GFP, CADM3-GFP, and E-cadherin-GFP in four separate 6-cm dishes. After 24 h, the cells transfected with CADM4-FLAG were co-cultured with cells transfected with CADM1-GFP, CADM3-GFP, or E-cadherin-GFP in 10-cm dishes at a 1:1 ratio. After 2-day culture, the co-cultured cells were treated with 1 mM DTSSP/PBS for 30 min at room temperature, and any cross-linking was quenched with 20 mM Tris-HCl (pH 7.4). The cells were lysed with TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, pH 7.8) containing a protease inhibitor cocktail (200 μM AEBSF, 10 μM leupeptin, and 1 μM pepstatin A) for 30 min at 4°C. The lysates were centrifuged at 12,000 rpm for 20 min at 4°C and the supernatants were immunoprecipitated using anti-FLAG M2 Affinity Gel (Sigma-Aldrich) overnight at 4°C. After washing the gel four times with TNE buffer, the FLAG-tagged CADM4 was eluted using 150 μg/mL of 3 × FLAG peptide (ApexBio, Houston, TX, USA) and subjected to Western blotting. The CADM1-GFP, E-cadherin-GFP, and CADM4-FLAG expression vectors were previously described (Kuramochi et al., 2001 (link); Williams et al., 2006 (link); Sakurai-Yageta et al., 2015 (link)). The CADM3-GFP expression vector was obtained by cloning CADM3 cDNA into the pEGFP-N3 BglII-SalI site (TaKaRa Bio Inc.).