Novablot system

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.

Cells seeded (10,000 cells/mL) and treated in 90 mm² Petri dishes were
harvested using 1 mM EDTA-PBS. Harvested cells were homogenized in RIPA buffer
(50 mM Tris, 150 mM NaCl, 0.5% Sodium deoxycholate, 0.1% SDS, 1.0% NP-40).
Supernatant was collected and protein content was estimated by Bradford method
spectrophotometrically. 30 μg Protein samples were then resolved in 10% and 7%
SDS-PAGE gels, transferred to PVDF membrane (Hybond-P GE healthcare, USA) using
the semidry Novablot system (Amersham Pharmacia, USA). Blots were probed with
primary mouse monoclonal antibodies anti-α-Tubulin (1:5000), anti-GFAP (1:3000),
anti-HSP70 (1:2500), anti-Mortalin (1:1000), anti-MAP-2 (1:2500) and anti-NCAM
(1:2000) diluted in 5% skimmed milk for overnight at 4 °C. Immunoreactive
protein bands were visualized after incubation with goat anti-mouse HRP
conjugated secondary antibody (Merck Millipore, USA) for 2 hours at room
temperature and development of blot with ECL Western blot detection system (GE
Healthcare, USA). Blots were developed and antibody labeling intensity was
quantified by using Image Quant LAS 4000 (GE Healthcare, USA).