Anti creb5

About 90% adherent cells were lysed with cold cell lysis buffer or 30 min at 4 °C and centrifuged at 12,000 rpm for 20 min at 4 °C. Protein supernatant was incubated with specific antibody (anti-CREB5: Santa Cruz, sc-130435; anti-ATF2: Immunoway, YT0382) or IgG control overnight at 4 °C. Subsequently, protein A/G agarose beads were added to the immunoprecipitation mixture with gentle rocking for 2 h at 4 °C. After washing with washing buffer, the beads with the immunoprecipitates were incubated with 1 × sample loading buffer and boiled for 10 min for subsequent western blot analysis.

Naringenin (Nar) (≥ 95%, N5893), streptozotocin (STZ) were purchased from Sigma (Sigma Aldrich, United States). Anti-PCNA (1:1000, ab29) and anti-OPN (1:500, ab63856) were purchased from Abcam. Anti-MMP2 (1:1000, bs-4605R) and anti-MMP9 (1:1000, bsm-54040R) were obtained from Bioss Biotechnology Co., LTD. (Beijing, China). Anti-α-SMA (1:1000, BM0002), anti-VEGFA (1:1000, BA0407) and anti-VEGFR2/KDR (1:1000, A00901-3) were purchased from Boster Biological Technology Co. Ltd. (Shanghai, China). Anti-CREB5 (1:1000, G420) was purchased from Santa cruz. The following antibodies: PI3K (1:1000, 4249), p-PI3K (1:500, 17366), Akt-pan (1:1000, 4685), p-Akt (Ser 473) (1:1000, 4060), Src (1:1000, 36D10), and p-Src (1:1000, D49G4) were obtained from CST. EdU Imaging kit was purchased from APE × Bio (United States). Trypsin, Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO.

Protein was extracted from cells or tissues by using RIPA buffer and quantified by the BCA method. Subsequently, protein was electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membranes followed by incubation with 5% nonfat powdered milk in Tris‐buffered saline at room temperature for 1 h. Then, the membranes were incubated with the specific primary antibodies overnight at 4 °C. The next day, after washed with TBST, membranes were incubated with secondary antibodies at room temperature for 1 h and washed again. The immunoreactive bands were visualized by an enhanced chemiluminescence system (Millipore, Billerica, MA, USA). The primary antibodies were as follows: anti-CREB5 (Santa Cruz, sc-130435), anti-ATF2 (Immunoway, YT0382) antibodies. GAPDH (Proteintech, 60004-1-Ig) were used as an internal control.