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. 2021 Jul 15;32(15):1347–1358. doi: 10.1091/mbc.E20-07-0470

FIGURE 3:

FIGURE 3:

ZO-2 modulates the effect exerted by PKC δ on TEAD nuclear accumulation. (A) Confluent monolayers of parental and ZO-2 KD MDCK cells were treated with 10 μM H89 to inhibit PKA, 27 nM Ro 31-8220 to inhibit cPKC α, γ, and β1 and nPKCε, 10 nM Gö-6986 to inhibit cPKC α, β, and γ and nPKCδ, or 0.1% DMSO as vehicle. Cytoplasmic and nuclear fractions were isolated and run in a SDS–PAGE and blotted with antibodies against ZO-2 and TEAD. GAPDH and lamin B1 were employed as loading controls for cytoplasmic and nuclear fractions, respectively. Left, representative images of Western blots from three independent experiments; right, densitometric analysis. Statistics done with one-way ANOVA followed by Fisher’s least significant difference test, *p < 0.05; **p < 0.01; ***p < 0.001. Each bar shows the mean ± SEM. (B) Schematic representation showing that in ZO-2 KD MDCK cells Ro-31822 blocks the nuclear exportation of TEAD mediated by nPKCε. (C) Schematic representation depicting in parental cells ZO-2 entry to the nucleus triggered by the inhibition of PKA with 10 μM H89. (D) Parental MDCK cells were transfected with a nPKCδ construct, and 24 h later nuclear fractions were obtained and processed for Western blot employing antibodies against ZO-2 and TEAD. Lamin B1 was employed as loading control of nuclear fractions. Left, representative image of Western blots from three independent experiments; right, densitometric analysis. Statistics done with Students’ t test, **p < 0.01. Each bar shows the mean ± SEM. (E) MDCK ZO-2 KD cells were transfected with a nPKCδ construct, and 24 h later nuclear fractions were obtained and processed for Western blot employing antibodies against TEAD. Lamin B1 was employed as loading control of nuclear fractions. Left, representative image of Western blots from three independent experiments; right, densitometric analysis. Statistics done with Students’ t test, n.s., nonsignificant. Each bar shows the mean ± SEM. (F) Cartoon showing that nPKCδ blocks ZO-2-TEAD cytoplasmic interaction and nuclear importation.