Bid associates with TRAF6 in microglia and astrocytes, as shown by coimmunoprecipitation and PLA. A, Coimmunoprecipitation of Bid and TRAF6 in BV-2 cells.

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Bid associates with TRAF6 in microglia and astrocytes, as shown by coimmunoprecipitation and PLA. A, Coimmunoprecipitation of Bid and TRAF6 in BV-2 cells. Bid associates with TRAF6 in microglia and astrocytes, as shown by coimmunoprecipitation and PLA. A, Coimmunoprecipitation of Bid and TRAF6 in BV-2 cells. BV-2 cells were stimulated with LPS (1 µg/ml) for 15 min or 1 h and Bid was immunoprecipitated. Negative controls included anti-Bid immunoprecipitation from bid-deficient mixed glia lysates, and IgG immunoprecipitation from all samples. Cells were lysed in RIPA buffer and analyzed for TRAF6 content after immunoprecipitation of Bid. B, Coimmunoprecipitation of Bid and TRAF6 in BV-2 cells overexpressing TRAF6-FLAG. BV-2 cells were stimulated with LPS (1 µg/ml) and lysed in RIPA buffer. FLAG was detected by Western blotting and represents TRAF6FLAG immunoprecipitated with Bid in BV-2 cells. An IgG immunoprecipitation was included as a negative control. C, Coimmunoprecipitation of Bid and TRAF6 in WT and bid-deficient primary mixed glia stimulated with LPS for 1 and 4 h (100 ng/ml). Samples were lysed post-LPS stimulation and Bid was immunoprecipitated from the lysates. The samples were analyzed for TRAF6 content by Western blotting. IgG immunoprecipitation was carried out as an additional negative control. D, Coimmunoprecipitation of Bid and TRAF6 in wild-type and bid−/− astrocytes. Purified astrocytes were stimulated with LPS (100 ng/ml) for 1 and 4 h, and lysed in RIPA buffer for Bid immunoprecipitation. The samples were analyzed for TRAF6 by Western blotting. E, Representative images of PLA and phase contrast in TRAF6-FLAG overexpressing BV-2 cells immunostained with anti-Bid and anti-TRAF6 (n = 2 wells/condition, 4 fields of view per well). Negative control representative images of PLA in TRAF6-FLAG overexpressing BV-2 cells immunostained with anti-TRAF6 and anti-HA-tag (n = 1 well/condition, 6 fields of view-LPS, 1 field of view + LPS), or immunostained with anti-Bid and anti-IRF2 (n = 1 well/condition, 7 fields of view). Scale bar, 10 µm. F, Quantification of PLA interactions in BV-2 cells. BV-2 cells were transfected with TRAF6-FLAG or empty FLAG vector and stimulated with LPS for 1 h. The cells were fixed with 3% paraformaldehyde, incubated with anti-Bid and anti-TRAF6 and PLA was quantified (significant increase of PLA dots in TRAF6 transfected versus control transfected cells and vehicle vs LPS treated cells, two-way ANOVA). Negative controls included immunostaining with anti-Bid plus anti-IRF2, and anti-TRAF6 plus anti-HA-tag. Sinéad Kinsella et al. eneuro 2016;3:ENEURO.0099-15.2016 ©2016 by Society for Neuroscience