Fig. 5. Expression of claudin-2, but not claudin-4, in MDCK monolayers increases the number of pores. (A) Representative immunoblots from cells used for TER and P_app studies demonstrate at least fourfold higher levels of claudin after induction (i) compared with those in uninduced (u) cells; claudin-2 expression in knockdown (KD) cells was approximately 2-10% of control cell values; occludin (occ) levels are unchanged. Approximate molecular masses: occludin, 60 kDa; claudin-2, 22 kDa, claudin-7, 20 kDa; claudin-4, 17 kDa. (B) Induction (filled bars) of claudin-2 compared with uninduced (unfilled bars) MDCK II monolayers results in a small increase in TER, and a large drop in C7 cells. Induction (filled bars) of claudin-4 in MDCK II monolayers causes a twofold increase in TER relative to uninduced (unfilled bars) monolayers; knockdown of claudin-2 results in a threefold increase in TER (filled bars) over parental cell values (unfilled bars). (C) Induction of claudin-2 (unfilled circles) in MDCK II monolayers results in a significant increase in the P_app specifically for PEG sizes that are of radius <4 Å, compared with uninduced monolayers (filled circles). Means and s.e. of four separate clones. (D) Corrected P_app of the 3.5 Å PEG₅ species reveals increases in pore number after inducing claudin-2 (filled bars) relative to uninduced (unfilled bars) in both MDCK II and MDCK C7 cells, but no change in MDCK II cells expressing claudin-4 or knockdown cells with decreased claudin-2 expression (means and s.e. from at least four determinations). (E) [³H]-Mannitol flux [disintegrations per minute (DPM) versus time] is unaffected by induction of claudin-2 (unfilled circles) compared with uninduced (filled circles) MDCK II monolayers.