Fitc 1

The C-ICD and C-ICDmut peptides were synthesized by Fmoc chemistry using the PSSM-8 automated peptide synthesizer (Shimadzu, Kyoto, Japan) and purified by reverse-phase high-performance liquid chromatography on a C18 column with a linear gradient from 0 to 90 % CH₃CN in 0.1 % trifluoroacetic acid for 60 min at a flow rate of 1 ml/min. The purified peptides were digested with trypsin and separated using the Smart System (GE Healthcare, Little Chalfont, UK) on a RP300 column with a linear gradient from 0 to 90 % CH₃CN in 0.1 % trifluoroacetic acid for 50 min at a flow rate of 1 ml/min, and the fractions were collected. The individual fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation tandem mass spectroscopy using the Axima Performance system (Shimadzu), and their amino acid sequences were confirmed by the 492HT protein sequencer (Applied Biosystems, Foster City, CA, USA). FITC-I (Dojindo) was used to label the peptides according to the manufacturer’s instructions. Briefly, the peptide and FITC-I were mixed at a weight ratio of 10:1 in phosphate-buffered saline (PBS) containing 2.5 mM carbonate and were reacted at 4 °C for 4 h. The FITC-labeled peptide was washed with PBS by five centrifugation cycles using an Amicon Ultra-10 K membrane filter (Millipore, Billerica, MA, USA).

Sacran was extracted from Aphanothece sacrum according to previous work^{31 (link)}. Xanthan gum extracted from Xanthomonas campestris was purchased from Taiyo Kagaku Co., Japan. FITC-I was purchased from Dojindo Molecular Technologies Inc., Japan. Top-side-open cells were prepared with two glass slides and a 1-mm-thick silicon spacer.