Bs 40296g hrp

The following primary antibodies were used: anti-CP110 (Proteintech, 2780–1-AP, 1:2000 for western blotting, 1:500 for immunostaining), anti-CEP97 (Proteintech, 22,050–1-AP, 1:1000), anti-turboGFP (Origene, TA150041, 1:2000 for western blotting or Proteintech, tbfms, 1:500 for immunostaining), anti-myc-tag (Invitrogen, MA1-21,316, 1:2000 for western blotting, 1:500 for immunostaining), anti-His-tag (Immunoway, YM3004, 1:2000); anti-acetylated α-tubulin (Sigma, T6793, 1:500), anti-γ-tubulin (Sigma, T6557, 1:500), EP1332Y (Abcam, ab52866, 1:4000), GT335 (Adipogen, AG-20B-0020, 1:4000 for western blotting, 1:500 for immunostaining), PolyE (Adipogen, AG-25B-0030, 1:4000 for western blotting, 1:500 for immunostaining), anti-ARL13B (Proteintech, 17,711–1-AP, 1:500), anti-β-actin (Sungene biotech, KR9001T, 1:4000). The secondary antibodies were goat-anti-mouse or donkey-anti-rabbit antibodies coupled with horseadish peroxidase (Bioss, bs-40296G-HRP and bs-0295D-HRP 1:4000) for western blotting or those coupled with Alexa Fluor 488 or 568 for immunofluorescence analysis (Invitrogen, A32723 and A-11011 1:750).

After tumor tissues were formalin-fixed and paraffin-embedded, they were sliced into 4-μm thick sections for HE (hematoxylin-eosin) staining and Immunohistochemical staining (IHC) for Ki67 (GB111499,1:200, Servicebio, Wuhan, China), P16-INK4A (10883-1-AP, 1:100, Proteintech, Wuhan, China) and P21 (YT3794, 1:50, Immunoway, Jiangsu, China). HE staining was performed using HE staining kit (C0105S, Beyotime). Besides, sections were subjected to deparaffinization, rehydration and antigen retrieval. After blocking, these samples were incubated with primary antibody overnight at 4°C. Subsequently, the slides were covered with HRP-conjugated secondary antibody goat anti-rabbit IgG(H+L) HRP (GAR0072, 1:200, Liankebio, Huangzhou, China) or goat anti-mouse IgG(H+L) (bs-40296G-HRP, 1:200, Bioss, Beijing, China) and then stained with diaminobenzidine (DAB) chromogen kit. Images were captured using an inverted microscope (Eclipse Ci-L, Nikon, Japan). The staining results were analyzed using Image-pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) and quantified by integrated optical density (IOD).

Western blot was used to detect protein levels of corresponding genes in 27 pairs of tumor and matched adjacent normal sample from our center. In brief, total protein was extracted using a lysate mixture containing RIPA and protease inhibitors. After concentration determination, an equal amount of total proteins from each sample was used for polyacrylamide gel electrophoresis. After transfer to PVDF membranes, the membranes were blocked and incubated with the primary antibody overnight at 4°C. After incubated with the secondary antibody at room temperature for 1 hour, ECL was added for exposure and development.
Primary antibodies used in this study include rabbit anti-β-tubulin (Abcam, ab6046, 1:1000 dilution for WB), rabbit anti-EPHX4 (Abcam, ab183739, 1:1000 dilution for WB), mouse anti-HES6 (Abcam, ab172800, 1:1000 dilution for WB), rabbit anti-NKD2 (CST, 2073T, 1:1000 dilution for WB), rabbit anti-OLR1 (Bioss, bs-2044R, 1:1000 dilution for WB) and anti-ONECUT2 (Bioss, bs-19643R, 1:1000 dilution for WB). A goat-anti-mouse-HRP (Bioss, bs-40296G-HRP, 1:10000 dilution for WB) antibody (Bioss, bs-19643R, 1:1000 dilution for WB) and a goat-anti-rabbit-HRP antibody (Bioss, bs-80295G-HRP, 1:10000 dilution for WB) were used in WB. The relative protein expression of the 5 genes in 27 GIAC patients (matched adjacent normal and tumor samples) were showed in Supplementary Table 6.