Itga11

Indomethacin, iRGD, and cilengitide were purchased from MCE (Junction, NJ, USA). MG132 and cyclohexamide (CHX) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). The ITGAV, fibronectin, ubiquitin, integrin β3 (ITGB3), p‐SMAD2 (S225), SMAD2, p‐SMAD3 (S423 and S425), and SMAD3 antibodies were obtained from Abcam (Cambridge, MA, USA). The p‐FAK (Tyr 925), FAK, PI3K p85α, p‐AKT (Ser 473), AKT, p‐GSK3β (S9), GSK3β, cyclin D1, CDK4, CDK6, SYVN1, NEDD4, ITCH, MYC, CBL, CBLB, anti‐Flag, and anti‐HA antibodies were obtained from CST (Beverly, MA, USA). The β‐Actin antibody was obtained from ZSGB (Beijing, China). The p‐PI3K p85α (Tyr 467), ITGA2, ITGA3, ITGA4, ITGA6, ITGA11, ITGB1, ITGB5, ITGB6, and ITGB8 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Ki67 antibody was obtained from Thermo Scientific (Waltham, MA, USA). Detailed information regarding the antibodies used within this study can be found in the Supplementary data. Plasmids encoding flag‐ITGAV and HA‐ubiquitin were obtained from Addgene (Watertown, MA, USA). Plasmids encoding HA‐SYVN1, MYC‐UB and MYC‐ITGB3 were from YouBio (Hunan, China). Lipofectamine 2000 was obtained from Invitrogen (Shanghai, China).

The tissues were processed routinely and stained with hematoxylin and eosin (HE) [39 (link)]. ITGA11 and Jab1 levels in the formalin-fixed, paraffin-embedded tissue were evaluated using immunohistochemical staining, as described in our previous work [40 (link)]. Briefly, the samples were sectioned and mounted on slides, following drying at 60°C for 1 hour. Slides were then deparaffinized in 2-xylene. To retrieval antigen, the slides were boiled for 3 minutes in 0.01 mol/L sodium citrate (pH 6.0) and then cooled at room temperature for 30 minutes. To block the Endogenous peroxidase activity, the slides were further immersed in 0.3% H₂O₂. Then the slides were incubated with the primary antibodies ITGA11 (Santa Cruz, sc-98740) and Jab1 (Santa Cruz, sc-13157) diluted at 1:200 overnight at 4°C and were detected by a secondary antibody kit (Dako Corp). ITGA11 and Jab1 expression were measured by counting no less than 400 tumor cells. Tumor cells were considered positive for markers when nuclear or cytoplasmic staining was present. The positivity represented the estimated fraction of positively stained cells (−, ≤5%; +, 5% to 25%; ++, 26% to 50%; +++, >50%). All experiments were performed in accordance with approved guidelines and regulations of Anyang Tumor Hospital.