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. Author manuscript; available in PMC: 2015 Sep 15.
Published in final edited form as: J Immunol. 2014 Aug 8;193(6):2850–2862. doi: 10.4049/jimmunol.1302778

Figure 4. Genetic or pharmacologic disruption of HDAC6 decreased phosphorylation but not acetylation of STAT3 in APCs.

Figure 4

(A) Non-target or HDAC6KD RAW264.7 macrophages were stimulated with LPS (1.0 μg/mL). After the indicated time points, cells were lysed and immunoblotted using specific antibodies against STAT3, acetylated-STAT3, phospho-STAT3 Y705, phospho-STAT3 S727, JAK2, phospho-JAK2, TYK2, phospho-TYK2 and GAPDH. (B) PEM were isolated from WT or HDAC6 KO mice and stimulated or not with LPS (1.0 μg/mL). After 24 hours, cells were lysed and immunoblotted using antibodies against HDAC6, tubulin, acetylated-tubulin, STAT3, pSTAT3 Y705, ac-STAT3 and GAPDH. (C) PEM from HDAC6 KO or WT mice were treated with LPS as in (B) and then subjected to ChIP analysis using α-STAT3 and α-p-STAT3 antibodies. (D) Non-target or HDAC6KD RAW264.7 cells were stimulated with IL-10 (10 ng/mL). After the indicated time points, cells were lysed and immunoblotted using specific antibodies as indicated on panel A. (E) PEM isolated from WT or HDAC6 KO mice were stimulated with IL-10 or left untreated. After 30 minutes, cells were lysed and immunoblotted using the indicated antibodies (F) PEM isolated from C57BL/6 mice were treated with Tub-A for 24 hours. Cells were then lysed and immunoblotted using the indicated antibodies. (G) PEM from HDAC6 KO as well as WT mice were stimulated with LPS (1.0 μg/ml) for 2 hr or remained untreated. Then, total RNA was isolated and analyzed by qRT-PCR for expression of Socs3, Vegf, Il1A and Il6 genes. GAPDH was used as control. The results are expressed as fold over non treated control cells normalized by GAPDH expression according to the Pfaffl equation(37). Three experiments were performed with similar results. (H) Production of IL-10 by non-target or STAT3KD macrophages in response to LPS. Data displayed (A-H) is from a representative experiment of three independent experiments with similar results.