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Fig. 3 | Cell & Bioscience

Fig. 3

From: Dysfunction in parkin aggravates inflammatory bone erosion by reinforcing osteoclast activity

Fig. 3

Parkin attenuates the acetylation of tubulin during osteoclast formation through the RANKL-dependent ERK pathway. A Mature osteoclasts (mOCs) from WT mice grown on glass coverslips were stained with Cy™3-conjugated antibodies (for β-tubulin; yellow) and FITC-conjugated antibodies (for parkin; green), counterstained for nuclei (DAPI, blue), and observed by confocal microscopy. B Cell lysates from mOCs were immunoprecipitated with an antibody recognizing parkin. Precipitated proteins and a 1% protein lysate input were analyzed using western blot analysis with anti-α-tubulin or -β-tubulin antibodies. Experiments were repeated at least three times with similar results. C mOCs of WT and Parkin−/− mice were grown on glass coverslips and stained using antibody DyLight™ 488-conjugated (for acetylated α-tubulin; green) and antibody DyLight™ 649-conjugated (for F-actin; red), counterstained for nuclei (DAPI, blue), and observed by confocal microscopy (left). Scale bar, 50 μm (left) and 20 μm (right). Fluorescence intensity of acetylated α-tubulin (Ac α-tubulin) was quantified (right). **P < 0.005 between the indicated groups. Data are represented as means ± SD from three independent experiments; P-values were calculated by the Kruskal–Wallis test. D Pre-fused OCs by M-CSF and RANKL for 2 days from WT and Parkin−/− BMMs were starved for 4 h and then retreated with RANKL at the indicated times, and the cells were analyzed by western blot. Representative data from three independent experiments are shown. E Pre-fused OCs from WT and Parkin−/− mice were starved for 4 h and then treated with RANKL in the presence of buffer, PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor). After 24 h, the cells were harvested and analyzed by western blot (upper), and their acetylated tubulin level was quantified (right). Data are represented as means ± SD from three independent experiments. **P < 0.005 and ***P < 0.001 vs. the WT group. P-values were calculated by Tukey post hoc comparison tests

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