Fig. 3

C-terminal lysines are required for HDAC6 inhibitor-induced accumulation of Cx32. a Mutation of C-terminal (bottom), but not cytoplasmic loop (top), lysines prevented Cx32 accumulation in response to TubA treatment. N2a cells transfected with either Cx32-Myc/6xHis or site-specific Cx32-Myc/6xHis K➔R acetylation-deficient mutants were treated with DMSO or TubA and processed for Western blot. Blots were probed for Myc-tag to detect Cx32, with beta-tubulin as a loading control. (Note that vertical lines between samples in figure indicate use of sister blots, run simultaneously.) (b) N2a cells were transfected with either Cx32-Myc/6xHis (WT), acetylation-deficient multi-site lysine to arginine mutant of all five cytoplasmic C-terminal lysines (5R), or acetylation-mimetic multi-site lysine to glutamine mutant of the five C-terminal lysines (5Q). WT, 5R, 5Q expressing cultures were treated with TubA or vehicle and assessed for Cx32 expression as in (a). c WT, 5R, 5Q expressing cultures were treated with TubA or vehicle and assessed for acetylation with anti-AcK. Cultures were immunoprecipitated with affinity purified rabbit-anti-Cx32 (E. Hertzberg) and probed with mouse monoclonal anti-Cx32 (7C6.7C; E. Hertzberg) or anti-AcK. Basal levels of acetylation were lower, as predicted, in untreated 5R and increased in 5Q relative to total Cx32. Treatment with TubA increased WT and 5Q acetylation, while 5R levels remained largely unchanged