Figure 5. MAK-V is not ubiquitinated by Nedd4.

(A) E2 enzyme specificity of ubiquitin-ligase activity co-purified with MAK-V-FLAG protein. MAK-V-FLAG protein purified from PC12TetOn cells was used in in vitro ubiquitination reactions containing E1 enzyme and indicated E2 ubiquitin-conjugating enzymes. Results of Western blotting with HRP-conjugated streptavidin to detect ubiquitinated proteins/polyubiquitin (marked by asterisk) are shown. The image was overlaid with an image of the same membrane that was consequently probed with anti-FLAG antibodies to detect MAK-V-FLAG protein (marked by arrows). (B) Depletion of Nedd4 does not affect stabilization of MAK-V-FLAG protein in cells in response to treatment with proteasome inhibitors. Parental PC12TetOn cells with inducible MAK-V FLAG expression (no miR) or their clonal derivatives expressing control microRNA (control miR) or microRNA to target Nedd4 transcript (Nedd4 miR) were treated with doxycycline to express MAK-V-FLAG protein and incubated with 100 µM of ALLN (ALLN) or 10 µM of MG132 (MG132) for 8 hrs or left untreated (-). Results of Western blot analysis of total cell lysates with anti-FLAG antibodies are shown (anti-FLAG). Nedd4 level was monitored with anti-Nedd4 antibodies (anti-Nedd4). MAK-V-FLAG protein marked with arrow, ubiquitinated higher molecular weight MAK-V-FLAG species marked by asterisk. Membrane was also probed with anti-α-tubulin antibodies (anti-α-tubulin) to monitor total protein loading. (C) MAK-V-FLAG protein and cytosols were prepared from parental PC12TetOn cells with inducible MAK-V-FLAG expression (Nedd4+) or its Nedd4-depleted clonal derivative expressing Nedd4 microRNA (Nedd4 -) and used in in vitro ubiquitination reaction containing E1 and UbcH5b E2 enzymes. Results of Western blot analysis with anti-FLAG antibodies are shown. Arrow marks products of MAK-V ubiquitination. Reactions in the presence of EDTA (EDTA +) were used as negative controls.