Fig. 4

Decrease in acetylation of α-tubulin is modulated by the interaction between parkin and HDAC6. A–C Schematic representation of full-length parkin protein and its deletion mutants binding to HDAC6. A Blue arrows indicated the binding region between parkin and HDAC6. B Parkin interacts with HDAC6. Empty vector, FLAG-tagged full-length parkin (1-465), Mt1 (1-217), Mt2 (1-310), or Mt3 (1-405) was co-transfected with GFP-HDAC6 in HEK 293 cells. Anti-FLAG immunoprecipitation was performed, followed by western blotting using anti-GFP antibodies. The input of FLAG for parkin, GFP for HDAC6, or β-actin is also shown. Red asterisk indicated overexpressed mutant form of parkin. C Interaction between parkin and HDAC6 is decreased by the Mt1 lacking RING1, IBR, and RING2 of parkin. Empty vector, FLAG-tagged full-length parkin, Mt1, Mt2, or Mt3 was co-transfected with GFP-HDAC6 in RAW 264.7 osteoclast precursor cells and was further cultured for 3 days (including M-CSF and RANKL), followed by western blotting using anti-acetylated α-tubulin, or anti-β-actin. D–F BMMs from WT mice were transfected with control- or HDAC6-specific siRNA and subsequently cultured with 30 ng/ml M-CSF and 10 ng/ml RANKL for 3 days. D Protein expression levels of HDAC6, acetylated α-tubulin, and cathepsin K were analyzed by western blot analysis. E Cathepsin K activity and F the number of resorption pits from the transfected cells were measured. Data represented as means ± SD from three independent experiments; *P < 0.05 and **P < 0.005 vs. the WT group. P-values were calculated by Tukey post hoc comparison tests