Mek1 2

After treatment with siRNA negative control (si-NC), si-Spry3, agomiR-NC, agomiR-18a, agomiR-BART10-5p, agomiR-18a combined agomiR-BART10-5p, antagomiR-NC, antagomiR-18a, antagomiR-BART10-5p, and antagomiR-18a combined antagomiR-BART10-5p, HONE1 and HONE1-EBV cells were harvested and lysed in lysis buffer supplemented with protease inhibitors. MicroRNA mimics, agomiRs, inhibitors, and antagomiRs were transfected at 50 nmol/L with Lipofectamine 2000 reagent (Invitrogen, USA). Protein lysate was resolved on 10% SDS-PAGE followed by blot transfer onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using the semidry NovaBlot system (Amersham Pharmacia, UK) for western blot analysis. After that, the membranes were first incubated with antibodies against GAPDH (Proteintech, USA), Spry3, Ras, c-Raf, MEK1/2, Erk1/2, mTOR, eIF4E1, VEGF, mmp2, and HIF1-α (Abcam, UK) overnight at 4°C, followed by a 1- to 2-h incubation with horseradish peroxidase-conjugated secondary antibody. The protein signals were detected using an enhanced chemiluminescence kit (Fdbio Science, China) and analyzed using the Bio-Rad (USA) imaging system and the associated software according to the manufacturer’s instructions. Antibodies concentrations are listed in Table S1.

The cells were plated and allowed to adhere in complete medium overnight, followed by treatment with the indicated reagent. The cells were then lysed in RIPA buffer containing protease and phosphatase inhibitors (Calbiochem, Darmstadt, Germany). Protein lysates were harvested and centrifuged, and the supernatants were collected and quantified with a BCA protein assay (Pierce Chemical Co., USA). Equal amounts of protein sample (20 µg) were subjected to SDS-PAGE analysis and electrophoretically transferred to nitrocellulose membranes (Millipore, Bedford, USA) using a Bio-Rad semidry transfer system. Protein expression was analysed using an ODYSSEY Infrared Imaging System (LI-COR Biosciences, Lincoln, USA). The primary antibodies included phospho-MEK (Ser217/221) PARP (Cell Signaling Technology, USA), MEK1/2, ERK1/2, phospho-p44/42 MAPK (ERK) (Thr202/Tyr204), c-Myc, bax, cyclin D1 (Abcam, UK), and GAPDH (Santa Cruz, USA).

Total protein was extracted using cold radioimmunoprecipitation assay (RIPA) buffer with proteinase inhibitors and phosphatase inhibitors. The protein was quantified by BCA assay kit (Beyotime Biotechnology, Beijing, China), separated on SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the PVDF membranes were incubated with the indicated antibodies as follows: MAP3K9 (Abcam), MEK1/2 (Abcam), pMEK1/2 (Abcam), ERK1/2 (Cell Signaling Technology), pERK1/2 (CST), NF-κB (p65, CST), and β-actin (Santa Cruz Biotechnology).