Par-3 mediates the inhibition of LIM kinase 2 to regulate cofilin phosphorylation and tight junction assembly -- Data Supplement - JCB--Chen et al. -- The Journal of Cell Biology

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Figure S1. Effects of suppressing E-cadherin, occludin, and Tiam1 on phospho-cofilin levels and effects of cofilin KD on tight junction formation. (A) Par-3 KD in MDCK cells increases phospho-cofilin levels. MDCK cells were transfected with control construct or three pSUPER constructs targeting different sequences of canine Par-3 mRNA (Par-3 KD). Total cell lysates were analyzed for total and phospho-cofilin. (B) Effects of E-cadherin and occludin KD on cofilin phosphorylation. MDCK cells were transfected with different pSUPER constructs to KD Par-3, E-cadherin, or occludin. Total cell lysates were analyzed with indicated antibodies. (C) Par-3 and E-cadherin KD have additive effects on phospho-cofilin levels. MDCK cells were transfected with indicated constructs to KD Par-3 or E-cadherin alone or together (double KD). Total cell lysates were analyzed with the indicated antibodies. Numbers indicate relative levels of phospho-cofilin normalized against the total cofilin level. (D) Tiam1 is not involved in increasing phospho-cofilin levels after Par-3 depletion. MDCK cells were transfected with the indicated constructs to KD Par-3 alone or to double KD Par-3 and Tiam1. Double KD did not reduce the increased phospho-cofilin level. Expression of constitutively active Tiam1 C1199 did not elevate the phospho-cofilin level compared with control cells. Asterisk indicates Tiam1 C1199. (E) KD of endogenous cofilin in MDCK cells. MDCK cells were transfected with control construct or four pSUPER constructs targeting different sequences of canine cofilin-1 mRNA (cofilin KD). Total cell lysates were analyzed for cofilin and occludin. (F) MDCK cells were transfected with control construct, pSUPER-cofilin #1 (cofilin KD (A)) or pSUPER-cofilin #1 + #4 (cofilin KD (B)). Cells were subjected to calcium switch and stained for occludin at various times after calcium readdition. (G) Western blot analysis of total cell lysates from F. The levels of actin-depolymerizing factor (ADF), a closely related protein to cofilin, were largely unaffected by cofilin KD. (H) MDCK cells were transfected with the indicated constructs, and the kinetics of TER establishment was monitored for 13 h after the readdition of calcium. Each value is the mean of triplicate measurements. n = 3. Error bars represent SD.