Golm1

The expression levels of GOLM1, E-cadherin, Vimentin, TGF-β1, Smad2, p-Smad2, and
GADPH were detected by Western blotting. First, equal amounts of proteins were
loaded onto 10% SDS-PAGE gels for separation and then transferred to
polyvinylidene fluoride membrane (Bio-Rad). Second, the membranes were blocked
with 5% nonfat milk for 2 h and then were incubated with primary antibodies, and
placed on shaker, at 4 °C overnight. Indicated primary antibodies in this study
were polyclonal rabbit antibodies against GOLM1 (1:1000 dilution; Affinity),
E-cadherin (1:5000 dilution; Proteintech), Vimentin (1:1000 dilution;
Proteintech), TGF-β1 (1:1000 dilution; Abclonal), Smad2 (1:1000 diluton;
Affinity), p-Smad2 (1:1000) and GADPH (1:1000 dilution; Affinity). Third, the
membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies
(1:600 dilution; Beyotime Biotechnology) after washing with TBST buffer.
Finally, the protein levels were detected by using an enhanced chemiluminescence
system (ECL Kit, Pierce Biotechnology) and captured on light-sensitive Xrayfilm
(Carestream). The optical densities of bands were analyzed by ImageJ
software.

Serial sections with 5 µm were cut from the tissue blocks, deparaffinized by
xylene, and hydrated in a series of graded alcohol. Then the sections were
incubated with specific antibodies for GOLM1 (rabbit polyclonal, 1:50,
affinity), E-cadherin (rabbit polyclonal, 1:100, Proteintech). Finally, sections
were stained by using the DAB chromogenic agent (Dako Corp). Negative control
experiments were routinely conducted. The slides were scored by 2 experienced
pathologists independently in the condition of unawareness of slides source.