FIGURE 8.

Roles of the ubiquitin-binding domain of SQSTM1 and HDAC6. A, schematic diagram of SQSTM1, HDAC6, and their mutant constructs. CAT, deacetylase catalytic domain. B, immunoblot analysis of interaction of MyD88 with SQSTM1, HDAC6, and their mutant proteins. 293T cells were transfected with FLAG-MyD88 together with HA-SQSTM1, HA-SQSTM1 ΔUBA, HA-HDAC6, enzyme-inactive HA-HDAC6, or HA-HDAC6 ΔBUZ. Immunoprecipitation (IP) with anti-FLAG-agarose was carried out with clarified cell lysates, followed by immunoblotting (IB) with anti-FLAG and anti-HA antibodies. n.s., nonspecific bands. C, quantification of the percentage of RAW264.7 cells stably expressing FLAG-MyD88-GyrB with condensed structures. Cells were transfected with HA-SQSTM1, HA-SQSTM1 ΔUBA, HA-HDAC6, enzyme-inactive HA-HDAC6, or HA-HDAC6 ΔBUZ. After 24 h, cells were stimulated with 0.1 μm coumermycin for 30 min. Immunofluorescent staining of cells was carried out with anti-FLAG and Alexa 488 anti-mouse IgG antibodies. Results, shown as the mean ± S.E. (error bars) (n = 3), were obtained from three images of microscopic fields including at least 30 cells and are representative of three independent experiments. *, p < 0.01 for comparison with the empty vector group.