Figure 7

Tyrosine-phosphorylated protein levels in V4 cells increased after treatment with S1-EVs in DMEM during cell growth. (A) Diagram of preparing cells to obtain lysates during cell growth in DMEM containing FBS in the presence or absence of S1-EVs at various time points after plating. (B) After detaching with 0.02% EDTA/PBS, GD2- V4 cells (5 × 10⁵) were placed in 6 cm dishes with regular medium for 24 h at 37 °C. Cells were washed with plain DMEM, and starved in serum-free DMEM at 37 °C for 2 h. Then cells were cultured in the presence or absence of S1-derived EVs (4 µg) in serum + DMEM, and incubated for 0, 5, 15, 30 and 60 min at 37 °C. After incubation, the cells were lysed, and the lysates were used for SDS‒PAGE. Subsequently, immunoblotting was performed with the anti-phosphotyrosine mAb PY20. (C) Band intensities (130 and 67 kDa) in (B) were measured and plotted using ImageJ (1.53t). (D) Lysates as mentioned in (B) were subjected to SDS‒PAGE and immunoblotted with individual anti-FAK mAbs (p-397FAK, p-576FAK, p-925FAK mAbs). Band intensities were measured by ImageJ (1.53t) and plotted in (E).