Figure 2

Snail expression downregulates components of the Crumbs complex and strongly represses Crumbs3 transcription. Two control and four Snail-FLAG clonal cell lines were cultured, harvested in denaturing buffer and 20 μg protein samples resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and immuno-blotted for the Par and Crumbs complexes as described (Straight et al., 2006). (a) Blots probed for Snail expression, (b and c) Par complex (Par3, PKCζ asterisk denotes a cross-reactive band), (d and e) Crumbs complex (PATJ, PALS1, Crumbs3). Actin immunoblots are shown as loading controls. (f) Cytosolic RNA was isolated from two control and four Snail-FLAG clonal cell lines and subjected to quantitative reverse transcription–PCR (qRT–PCR) to detect levels of Crumbs complex transcripts. Bar graph depicts the mean transcript levels from three experiments (error bars indicate s.e.m.). Results were normalized to glycer-aldehyde-3-phosphate dehydrogenase (GAPDH). The qRT-PCR method was previously described (Straight et al., 2006). Additional antibodies used: polyclonal Snail E-18 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); polyclonal actin (Sigma, St Louis, MO, USA); horseradish-peroxidase-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, England).