Fig 2: LynΔN is degraded by the N-end rule pathway(a) Wild type LynΔN and valine-LynΔN were transfected into HEK293T. After transfection the cells were treated with 100 μg/ml cycloheximide (CHX) to block protein synthesis. At the indicated time points, cells were lysed and resolved by SDS-PAGE. The amount of LynΔN protein remaining was visualized by WB analysis with an anti-FLAG M2 antibody Loading control analysis was done by WB analysis using an anti-actin antibody. (b) LynΔN with N-terminal arginine (type I destabilizing N-termini) and methionine (stable N-termini) were transfected into HEK293T and analyzed as in (a). (c) Stability of LynΔN was visualized in HEK293T cells in the presence and absence of 10 μM MG132 and analyzed as in (a). (d) Stability of LynΔN was visualized in HEK293T cells that were also transfected with a plasmid mixture expressing shRNAs targeting UBR1 and UBR2 or controls. Both vector control and plasmids expressing scrambled shRNA sequences were used as controls. In all cases the total amount of plasmid DNA was used for each transfection was kept constant. (e) Knock down of UBR1 after shRNA treatment in (d) was verified by WB analysis for endogenous UBR1 after SDS-PAGE on a 5% gel. (f) Knock down of UBR2 after shRNA treatment in (d) was verified by WB analysis for endogenous UBR2 after SDS-PAGE on a 5% gel. (g) Stability of LynΔN was compared to the stability of the inactive K275R-LynΔN mutant.