Fig. 2

Identification of the RanBPM and HDAC6 domains that mediate their association. a Schematic representation of HA-tagged RanBPM wildtype (WT) and deletion mutant constructs ΔLisH and ΔCTLH. b Left, whole cell extracts were prepared from RanBPM shRNA Hela cells untransfected (−) or transfected with HA-RanBPM-WT, HA-RanBPM-ΔLisH, or HA-RanBPM-ΔCTLH constructs. RanBPM was immunoprecipitated with an HA antibody and immunoprecipitates were analyzed by western blot with HDAC6 and RanBPM antibodies. Input, 5% input extract. Right, quantification of relative amounts of co-immunoprecipitated HDAC6 normalized to immunoprecipitated RanBPM. Results are averaged from three different experiments with error bars indicating SEM. P < 0.05 (*). c Schematic representation of HDAC6 full length (FL) and deletion mutant constructs. d Left, whole cell extracts were prepared from HDAC6 knockout MEFs untransfected (−) or transfected with full length (FL) or ΔN439 HDAC6 constructs. HDAC6 was immunoprecipitated with a FLAG antibody and immunoprecipitates were analyzed by western blot with RanBPM and HDAC6 antibodies. Input, 5% input extract. Right, quantification of relative amounts of co-immunoprecipitated RanBPM normalized to immunoprecipitated HDAC6. Results are averaged from three different experiments with error bars indicating SEM. P < 0.05 (*). e Left, whole cell extracts were prepared from HDAC6 knockout MEFs untransfected (−) or transfected with HA-1-503 or HA-1-840 HDAC6 constructs and immunoprecipitation and analysis was performed as described above except that HDAC6 mutant immunoprecipitation was verified using an HA antibody. Arrows indicate the position of the HA-HDAC6 mutants. The asterisk indicates residual RanBPM signal from previous hybridization. Right, quantification of relative amounts of co-immunoprecipitated RanBPM normalized to immunoprecipitated HDAC6. Results are averaged from three different experiments with error bars indicating SEM. P < 0.05 (*)