Fig. 5.

(a) The anti-Ub protein conjugate western blots for the Met4 enriched samples from control and 0.1 mM Cd²⁺ treated cells and their corresponding total cell lysates are shown. The western blots of the Met4 captures show a decrease in overall ubiquitination of Met4 as a result of Cd²⁺ treatment. The western blots of the whole cell lysates on the other hand do not show a significant change in ubiquitination as a result of Cd²⁺ treatment; (b) the SRM assay shows that all ubiquitinated forms of Met4 in enriched samples including poly-ubiquitinated form via lysine 11, 48, and 63 decrease as a result of Cd²⁺ treatment while in the total cell lysates of the Cd²⁺ treated cells this trend is the opposite; (c) shows the link/total Ub ratio for all detected inter-Ub links. K11/total Ub ratio (representing poly-ubiquitination via K11) is only detectable in Met4 enriched sample before Cd²⁺ treatment. K48/total Ub ratio (representing poly-ubiquitination via K48) remains unchanged for the Met4 enriched samples and whole cell lysate after the Cd²⁺ treatment. In contrast, K63/total Ub ratio (representing poly-ubiquitination via K63) shows a decrease in Met4 enriched sample after the treatment and an increase in the whole cell lysate.