Mical1

Tumor specimens were obtained from Outdo biotech (Shanghai, China). Thirty primary human gastric tumor samples and their corresponding paracancerous tissue samples were used for immunohistological staining in our study. The sections were deparaffinized and rehydrated. Peroxidase blocking was done with 3% H₂O₂ in methanol for 15 min at 37°C. Antigen retrieval was performed by transferring the sections into EDTA buffer (pH 8.0). The sections were then blocked by goat serum and applied with MICAL1 (proteintech, Hubei, China) or NEDD9 (absin, Shanghai, China) antibody at 4°C overnight. Next, the sections were treated with the secondary antibody (MXB, Fujian, China) for 1 h at room temperature. After counterstaining with hematoxylin, the slides were mounted and photographs were obtained using an Olympus BX51 microscope. Reagents for immunohistochemistry were all obtained from ZSGB-BIO (Beijing, China). The Immuno Reactive Score (IRS) was calculated as intensity of the staining reaction multiplied by the percentage of the number of positive cells as previously described(Dumitru et al., 2013 (link); Medale-Giamarchi et al., 2013 (link)).

Subconfluent cells were washed with PBS and lysed for 20 minutes on ice in a RIPA buffer containing 1% protease inhibitor cocktail (Beyotime). The protein was collected, and its concentration was determined by the BCA protein assay reagent kit (Thermo Fisher Scientific) then separated by SDS‐PAGE and transferred to nitrocellulose membrane. The membrane was then blocked with 5% non‐fat milk for 1 hour at room temperature and incubated with primary antibody overnight at 4°C. The following antibodies were used: GAPDH (Bioworld), MICAL1 (Proteintech) (Santa Cruz), NF‐kB (Santa Cruz), β‐catenin, p‐β‐catenin, p‐GSK‐3β, p‐S6K, histone‐H3, ERK, p‐ERK, Akt and p‐Akt (Cell Signaling), cyclin D (ABclonal), c‐myc, CDK (2, 4, 6) and cyclin (A, E) (Santa Cruz). The membranes were incubated with secondary HRP‐conjugated antibodies (Santa Cruz) and visualized with ECL reagent (Millipore). Digital images of immunoblots were obtained with a Chemidoc XRS and analysed using the image analysis program Quantity One (Bio‐Rad).