Cell lysates

COS-7 were seeded in 6-well plates or 10 cm petri dishes and after 24 h incubation scraped with 100–500 µl lysis buffer (1% Triton X-100, 20 mM HEPES, pH 7.4, 130 mM NaCl, 10 mM NaF, 0.03% PIC) on ice. MDCKC7, Caco-2, MDCKII, MDCKII QKO, and MDCKII QKO FLAG-tagged or untagged Cldns expressing cells were seeded in 6-well plates and after 5–7 days scraped with 100 µl lysis buffer (1% Triton X-100, 20 mM HEPES, pH 7.4, 130 mM NaCl, 10 mM NaF, 0.03% PIC) on ice. The solutions were transferred into pre-cooled 1.5 ml vials and incubated for 30 min on ice. The protein-containing supernatant was isolated via centrifugation at 17,000 × g for 20 min at 4 °C. 1–10 µl of the supernatant was incubated with 490–499 µl H₂O and 500 µl 2x Bradford reagent for 5 min and the protein concentration was determined by measuring the OD595 with a photometer (BioPhotometer plus, Eppendorf). The protein lysates were denaturized with 6x Laemmli buffer (0.375 M Tris pH 6.8, 12% SDS, 60% glycerol, 0.6 M DTT, 0.06% bromophenol blue) for 5 min at 95 °C and stored at −20 °C.