Figure 1.

GA targets RAS and co-localizes with RAS in A549 cells. (A) Synthesis of alkynyl-modified GA (alkynyl-GA). (B) Synthesis of GA-modified functionalized MMs (probe 1) and the process for capturing and releasing the target protein. (C) Lane 1 shows A549 lysate as a loading control, Lane 2 shows the lysate captured by the azide-modified MMs as a negative control, and Lane 3 shows the lysate captured by probe 1. Markers indicate the molecular weight. The concentrations of all protein samples were adjusted to equal amounts before capture and were adjusted to the same volume after capture for SDS-PAGE and Western blot analyses. (D) Synthesis of fluorescent click product (probe 2). (E) Fluorescence intensity of the click product (probe 2) compared with alkynyl-GA, GA and N₃-tag. (F) Analysis of the co-localization of alkynyl-GA and the RAS protein using fluorescence confocal microscopy. The GA (10 µmol/L) treatment group showed little fluorescence. But obvious alkynyl-GA (1 µmol/L) fluorescence was observed in the cytoplasm (green). The specific fluorescence was competitively ablated by a 10 µmol/L GA treatment. Alexa Fluor594 staining for RAS (red) was found in the cytoplasm and membrane and partially co-localized with alkynyl-GA (yellow), as indicated by the arrows, scale bar 10 µm.