Siah2

Tissue sections were stained with antibodies to LATS2 (Novus, USA), SIAH2 (Novus, USA), CTGF (Santa-Cruz, USA), YAP (CST, USA) and α-SMA (Protein-tech, USA) for 14–16 h at 4℃ and followed by staining with Alexa-488 conjugated anti-Rabbit and Alexa-595 conjugated anti-Mouse respectively. 4’, 6-diamidino-2- phenylindole dihydrochloride (DAPI) was used to color nuclei. Confocal images were recorded by FV3000 confocal microscope (Olympus, Japan).

Cells were lysed and analyzed by Western blotting as described previously [24 (link)]. Primary antibodies used are as follows: Flag, α-tubulin, β-actin (Sigma-Aldrich); E-cadherin, N-cadherin (BD Laboratories); Siah1 (as previously described [44 (link)]); Siah2 (Novus Biologicals); Siah2, Zeb1 (Santa Cruz Biotechnology); Vimentin, Zeb1 (Cell Signaling Technology); HA (a kind gift from Rick Pearson, Peter MacCallum Cancer Centre). To quantify Western blots, densities of bands were measured in Image J [45 ]. These values were then normalized to the densities of their corresponding loading control. In cycloheximide treatment experiments, values of samples were then normalized to the positive control.