Figure 4.

Ubiquitination of proteins in R and T cells passaged four times in the presence or absence of tunicamycin. Panel (a) detection of P-gp in immunoprecipitates obtained with rabbit polyclonal anti-ubiquitin antibody (IgG). Whole cell lysates were immunoprecipitated, and subsequently P-gp was detected using Western-blotting with a c219 antibody. The detection of rabbit IgG heavy chain (50 kDa) using an anti-rabbit antibody was included as an internal standard; Panel (b)—detection of ubiquitinated material on the cell surface of R and T cells using a rabbit anti-ubiquitin polyclonal antibody and goat anti-rabbit antibody conjugated to Alexa Fluor 660 as primary and secondary antibodies, respectively; Panel (c) represents the same experiments as panel (b) but evaluated in InCell confocal image detection. Upper panels represent cell visualization in light microscopy mode, and the lower panels represent detection of Alexa Fluor 660 fluorescence. S represents sensitive cells cultivated four times in the absence of tunicamycin. R, T and R_T, T_T represent either R or T cells passaged four times in the absence or presence of tunicamycin (0.1 μmol·dm⁻³), respectively. Data in each panel are representative for three independent experiments.