Fig. 7. The RanBPM LisH/CTLH domain is required for aggresome formation and HDAC6 interaction.

(A) Schematic representations of HA-tagged RanBPM wildtype (WT) and deletion mutant constructs ΔN2, Δ212, Δ360 and ΔC4. The RanBPM conserved domains are indicated (CRA, CT11-RanBPM). (B) Hela cells stably expressing RanBPM shRNA were transfected with HA-WT, HA-ΔN2, HA-Δ212, HA-Δ360 and HA-ΔC4 and were fixed 16 h following 10 µM MG132 treatment. Cells were processed for immunostaining with antibodies to HA and HDAC6 and mounted with DAPI. At least 100 transfected cells were scored per experiment for the presence of aggresomes and the results are expressed as percentage of cells containing HDAC6 aggresomes. Results are averaged from four different experiments, with error bars indicating SE. P<0.05 (*). (C) Representative images of HA-tagged RanBPM constructs transfected into RanBPM shRNA Hela cells and treated with 10 µM MG132, processed as described above. (D) The RanBPM LisH/CTLH domain is necessary for interaction with HDAC6. Right, whole cell extracts were prepared from RanBPM shRNA Hela cells untransfected (–) or transfected with HA-WT-, HA-Δ360 or HA-Δ212 constructs. RanBPM was immunoprecipitated with a RanBPM antibody, and immunoprecipitates analyzed by western blot with an HDAC6 antibody. RanBPM WT and deletion mutant immunoprecipitation was verified using an HA antibody. Input, 5% input extract. Left, quantification of relative amounts of co-immunoprecipitated HDAC6 normalized to immunoprecipitated RanBPM. Results are averaged from three different experiments with error bars indicating SD. P<0.05 (*). Scale bar: 10 µm.