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. Author manuscript; available in PMC: 2017 Nov 1.
Published in final edited form as: Mol Neurobiol. 2015 Nov 13;53(9):6270–6287. doi: 10.1007/s12035-015-9537-z

Fig. 3.

Fig. 3

Parkin facilitates K63-linked polyubiquitination of mutant SOD1 in cells. a SH-SY5Y cells expressing indicated S-tagged SOD1 WT or mutants, Myc-tagged parkin, and HA-tagged ubiquitin were treated with vehicle (0.1 % DMSO) or 20 μM MG132 for 8 h. In vivo ubiquitination of SOD1 WT or mutants was assessed by S-protein bead pulldown under denaturing conditions followed by immunoblotting using anti-HA and anti-S-tag antibodies. Ubn, polyubiquitin. b In vivo ubiquitination analysis in SH-SY5Y cells expressing Myc-tagged SOD1 A4V, GFP-tagged parkin, and indicated HA-tagged ubiquitin WT or mutants following treatment with 20 μM MG132 for 8 h reveals that parkin preferentially promotes K63-linked polyubiquitination of SOD1 A4V. Asterisk denotes a nonspecific band. c In vivo ubiquitination analysis in SH-SY5Y cells expressing S-tagged SOD1 G93A, Myc-tagged parkin, and indicated HA-tagged ubiquitin WT or mutants following treatment with 20 μM MG132 for 8 h shows that parkin preferentially promotes K63-linked polyubiquitination of SOD1 G93A