Skip to main content
. Author manuscript; available in PMC: 2014 Sep 26.
Published in final edited form as: Mol Cell. 2013 Sep 12;51(6):819–828. doi: 10.1016/j.molcel.2013.08.016

Figure 3. HDAC6 binds unanchored ubiquitin chains during aggresome clearance in a Poh1-dependent manner.

Figure 3

(A) Representative image of HDAC6 and aggresome (ub) in Poh1 KD cells 24h after MG132 washout. (B) FLAG-HDAC6 was immuno-precipitated under the following conditions: 1) no treatment, 2) 5μM MG132 for 24h, 3) 12h MG132 washout, 4) 24h MG132 washout. The immune complexes were subjected to heating, and the eluates were treated with Isopeptidase T (IsoT) as indicated (see Methods for details). Samples were analyzed by immuno-blotting with antibodies for ubiquitin or HDAC6. Note that MG132 washout (conditions 2 to 3) led to a decrease in total ubiquitinated protein levels in whole cell lysate (WCL), but an increase in HDAC6-associated free ubiquitin chains. (C) Free ubiquitin chains associated with HDAC6 in wild type and Poh1 KD cells were assessed 12h after MG132 washout. Note that Poh1 KD led to the accumulation of total ubiquitinated proteins (left panel) but a reduction of HDAC6-associated unanchored ubiquitin chains (right panel). (D) Free ubiquitins chains released from HDAC6-IP components were immuno-blotted using a pan-ubiquitin antibody (pan-ub), K63-specific ubiquitin antibody (K63-ub) and K48-specific ubiquitin antibody (K48-ub) sequentially. Recombinant K63- or K48-linked poly-ubiquitin chains were loaded onto the same gel to validate the specificity of linkage-specific ubiquitin antibodies. See also Figure S5.