Anti six3

The details of these procedures were described previously [37 (link)]. Anti-SIX3 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-81985), anti-FLAG (Abcam, Cambridge, MA, USA; ab205606), anti-HA (Abcam; ab9110), and anti-IgG (Santa Cruz Biotechnology; sc-2027) antibodies were used for co-immunoprecipitation (Co-IP). Immunoprecipitates were washed at least five times and subjected to western blot analysis using anti-TRIM27 (Abcam; ab78393), anti-NEDD4 (Abcam; ab236512), anti-SMURF2 (Abcam; ab94483), anti-RNF6 (Abcam; ab204506), anti-SYVN1 (Abcam; ab170901), anti-MDM2 (Abcam; ab16895), and Anti-SIX3 (Abcam; ab172131) antibodies. For ubiquitination assays, the lysates of A549 cells transfected with siTRIM27-1 or siNC were used for IP with an anti-IgG (Santa Cruz Biotechnology; sc-2027) or Anti-SIX3 antibody (Santa Cruz Biotechnology; sc-81985) and Protein A/G PLUS-Agarose (Novex, Oslo, Norway), which was performed at 4° C overnight. The eluted proteins were then detected by western blot analysis using an anti-ubiquitin (Ub) antibody (Abcam, ab7780).

Equal amounts of protein samples were separated by 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Bands were probed immunologically using anti-Six3 (Santa Cruz), GSK‐3β (EnoGene), Phospho‐GSK‐3β (Ser9) (EnoGene), β‐catenin (EnoGene), Phospho-β‐catenin (Ser37) (EnoGene), Slug (EnoGene), E‐cadherin (EnoGene), N‐cadherin (EnoGene), c-Myc (EnoGene), and cyclin D1 (EnoGene). GAPDH (Cell signaling) was probed as an internal reference. Signals were detected using an enhanced chemiluminescence (ECL) system according to the manufacturer’s instructions.