Kinase activity of p110δ is required for TGN recruitment of Dyn2. (A and B) Unstimulated RAW264.7 macrophages (unstim) and macrophages activated by LPS for 2 h in the absence or presence of 5 µM IC87114 were fixed for immunostaining for PtdIns(3,4,5)P₃ (red) and TNF (green). DAPI (blue) labeling depicts nuclei. Appearance of PtdIns(3,4,5)P₃ upon activation at perinuclear Golgi membranes colocalizing with TNF. This PtdIns(3,4,5)P₃ detection was selectively lost in the presence of IC87114. (C) RAW264.7 cells transiently transfected with YFP-p230_GRIP (green) and untagged WT Dyn2 before activation by LPS for 2 h in the presence of DMSO (control) or 5 µM IC87114 were fixed for Dyn2 immunostaining (red). (B and C) Arrows indicate examples of colocalization of Dyn2 with p230 on TGN membranes that disappeared upon IC87114 treatment. (D) Activated cells coexpressing YFP-p230_GRIP and Dyn2 treated with DMSO (control) or IC87114 as described in C were analyzed by image analysis to quantify relative distribution intensities of Dyn2 (TGN vs. cytoplasm). Data are represented as mean ratio ± SEM from 30 cells over three transfections. ***, P < 0.0001. Insets show magnified views of boxed regions. Bars, 10 µm.