Dithiothreitol (dtt)

We modified the method reported by Walev et al. (2001) (link) to introduce GMNPs to cells. First, streptolysin O (SLO; Wako Pure Chemical) was activated by reduction with DTT (Nacalai Tesque). Stock solutions of SLO with a final concentration of ~5 U/μL were prepared.
HEK cells (5 × 10⁵) were rinsed with PBS(−) and resuspended in 20 μL of PEG-GMNP solution (NanoImmunotech) and 5 μL of 5× HBPS (1 mM Ca²⁺, 1 mM Mg²⁺). These cells were incubated in a 37°C incubator for 12 min in the presence of 1 μL of SLO. The pore-forming activities of SLO were inactivated by adding DMEM containing 10% FBS (0.5–1 mL) for 20 min or longer.

Samples immunoprecipitated with antibodies against decorin or periostin were subjected to SDS-PAGE, and the gels were stained using the Silver Quest Staining Kit (Invitrogen). The stained gel bands were cut out and treated with dithiothreitol (DTT, Nacalai Tesque, Kyoto, Japan) dissolved in ammonium hydrogen carbonate (Nacalai Tesque), followed by treatment with iodoacetamide (Wako, Osaka, Japan). After the gels were dried, 20 μl of 0.05 pmol/μl trypsin (AB SCIEX) solution was applied to each gel piece and incubated for 12–16 h at 37°C to digest proteins. Digested peptides were extracted by washing the gel pieces twice with 50% trifluoroacetic acid (TFA, Wako), followed by washing with 80% TFA. The purified peptide samples were injected onto a reversed-phase C18 column (HiQ sil C18W-3P, 3 μm, 120 Å; KYA TECH Corp.) and separated by nanoflow liquid chromatography (300 nL/min) on a nano LC Dina-A system (KYA TECH Corp.) in line with a Q-TRAP 5500 instrument (AB SCIEX) using a 75-min gradient of 5–100% acetonitrile in 0.1% formic acid.

Madin-Darby canine kidney (MDCK) II cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (Nichirei Biosciences, Tokyo) and 1% penicillin-streptomycin (Nacalai Tesque) in a humidified atmosphere containing 5% CO₂.
Capsaicin, FD4, and NEM were purchased from Sigma (St. Louis, MO). LatA was from Wako Pure Chemical Industries (Osaka, Japan). DTT was from Nacalai Tesque, and A-967079 was from Cayman (Ann Arbor, MI).
Antibodies to occludin (#71-1500), Zo-1 (#61-7300), and tricellulin (#700191) were purchased from Invitrogen (Grand Island, NY). Anti-claudin-1 (#13050-1-AP) and anti-cofilin (#3312) was purchased from Proteintech Group, Inc. (Rosemont, IL) and Cell Signaling Technology (Beverly, MA), respectively. Anti-β-actin (#125K4769) and anti-phospho-cofilin (Ser 3, #sc-21867-R) were from Sigma and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgGs were from Kirkegaard & Perry Laboratories (Gaithersburg, MD).
All other reagents were of reagent grade and purchased from Nacalai Tesque unless otherwise noted.