Bortezomib and P8 Modulation of Cellular Proteome

HeLa cells were seeded and cultured in 35 mm confocal dish and 25 cm² culture flask for fluorescence and gel analysis, respectively before treatment of Bortezomib (0.8 µm) and P8 (5 µm) for 24 h. For fluorescence analysis, cells were next fixed and permeabilized with 4% formaldehyde and 0.1% Triton X‐100 at room temperature consistently for 30 min. The cells were then added with propargylamine (10.0 mm) before white light illumination for 20 min. Freshly premixed click solution (1.7 mm TBTA, 50.0 mm CuSO₄, 50.0 mm TCEP, and 50.0 mm BODIPY‐N₃) was finally added to pretreated cells and reacted for 1 h. Cells were washed with PBS three times to remove unreacted click solution and treated with Hoechst 33342 before imaging with Olympus FV1000 FluoViewTM confocal microscope. For gel analysis, cells were incubated with propargylamine (10.0 mm) before white light illumination for 20 min. Next, cells were ultrasonic lysed with 1% SDS followed by reaction with newly prepared click solution (1.7 mm TBTA, 50.0 mm CuSO₄, 50.0 mm TCEP, and 50.0 mm TMR‐N₃). The obtained proteome samples were precipitated with acetone before gel analysis.