Ab81083

Total protein in cells was extracted by RIPA solution (GenePharma) and the protein concentration was estimated through BCA Protein Assay Kit (Beyotime). Equal amounts protein samples were loaded on each lane and separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Next, the membrane were cultivated with primary antibodies against PCNA (ab92552, 1:1,000 dilution, abcam), Ki-67 (ab245113, 1:10,000 dilution, abcam), Bax (ab81083, 1:1,000 dilution, abcam), Caspase-3 (218161, 1:1000 dilution, abcam) and GAPDH (ab181603, 1:1,000 dilution, abcam) at 4°C overnight. The membrane was washed with PBST for three times. Then the membrane was incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721, 1:1,000 dilution, abcam). The bands were visualized using a chemiluminescence detection kit (Beyotime).

The tissues were treated with 0.01 M citric acid for antigen retrieval. After washing with PBS, the slides were blocked with TBST containing 10 goat serum for 1 h. Then, the tissues were incubated with primary antibodies overnight at 4° C. The primary antibodies and dilution times were listed as follows. GAP43 (1:1000, ab232772, Abcam, UK), NF421 (1:800, ab187374, Abcam, UK), GFAP (1:1000, ab68428, Abcam, UK), Bax (1:800, ab81083, Abcam, UK), Cleaved Caspase-3 (1:1000, #961S, Cell signaling, USA), Bcl-2 (1:800, ab32124, Abcam, UK). Then, the sections were washed using PBS and incubated with related secondary antibodies for 2 h at room temperature. The secondary antibodies and dilution times were listed as follows. Goat anti-rabbit lgG (1:2000, ab205718, Abcam, UK) and Goat anti-mouse lgG (1:2000, ab205719, Abcam, UK). All sections were mounted and observed under a microscope (Leica, Germany). Image J software was used to quantify and normalize images.

Excised tumor and gastric tissues were fixed in 4% paraformaldehyde, dehydrated and embedded with paraffin, finally cut into tumor sections. Consecutive 4-μm-thick tumor sections were immunostained with specific primary antibodies targeting E-cadherin (ab76055), N-cadherin (ab202030), Bax (ab81083) and bcl-2 (ab196495) from Abcam as per the established protocol.