MAK-V is subjected to ubiquitin-dependent proteasomal degradation.

<p>(<b>A</b>) Doxycycline-treated (<i>DOX +</i>) or untreated (<i>DOX</i> -) PC12TetOn MAK-V-FLAG cells were incubated with 100 µM of ALLN (<i>ALLN</i> +) or vehicle (<i>ALLN</i> -) for 8 hrs. Results of Western blot analysis of total cell lysates with anti-FLAG antibodies are shown. MAK-V-FLAG protein marked with arrow, ubiquitinated higher molecular weight MAK-V-FLAG species marked by asterisk. To monitor total protein loading, membrane was re- probed with anti-α-tubulin antibodies. (<b>B</b>) HEK293 cells were transiently transfected with plasmid for MAK-V-FLAG protein expression and treated with 10 µM MG132 (<i>MG+</i>) for indicated time prior to lysis or left untreated (<i>MG-</i>). Lysates were blotted with anti-FLAG antibodies to detect MAK-V-FLAG protein (<i>anti-FLAG</i>). To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>C</b>) HEK293 cells were transfected with plasmid for HA-tagged ubiquitin expression alone (<i>MAK-V-FLAG -</i>) or together with plasmid for MAK-V-FLAG protein expression (<i>MAK-V-FLAG +</i>). Cells were treated with MG132 prior to lysis. MAK-V-FLAG protein was precipitated from lysates with anti-FLAG M2 affinity gel as described for PC12TetOn cells. Anti-FLAG antibodies were used to detect MAK-V-FLAG protein (<i>anti-FLAG</i>) in immunoprecipitates (<i>IP: anti-FLAG</i>), and ubiquitin was detected with anti-HA antibodies (<i>anti-HA</i>). To monitor HA-tagged ubiquitin and MAK-V-FLAG protein expression, aliquots of lysates were blotted as described above. To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>D</b>) HEK293 cells were co-transfected with MAK-V-FLAG expression plasmid and plasmid for expression of FLAG-tagged wild-type ubiquitin (<i>wt</i>), its K48R (<i>K48R</i>) or K63R (<i>K63R</i>) mutant. Results of Western blot analysis of total cell lysates with anti-MAK-V antibodies are shown. To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. To control variations in basal level of MAK-V-FLAG protein expression, samples were also probed with antibodies against neomycin phosphotransferase II (<i>anti-NPT</i>) which is encoded by MAK-V-FLAG expression vector.