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. 2014 Mar 25;24(5):576–594. doi: 10.1038/cr.2014.33

Figure 1.

Figure 1

Rab10 interacts with MARCKS. (A) Expression of Rab10 and MARCKS in the rat brain at different developmental stages. (B, C) Co-immunoprecipitation (co-IP) of Rab10 and MARCKS in homogenates of P0 rat brains. (D) Cortical neurons at DIV2 were treated with different concentrations of OA for 6 h. Cell lysates were subjected to immunoblotting (IB) with antibodies against MARCKS or phosphorylated MARCKS (pMARCKS), with GAPDH or actin as loading control. (E) Cell lysates from cultured cortical neurons treated with OA (100 nM, 6 h) or vehicle at DIV2 were subjected to pull-down with glutathione-sepharose beads coupled to GST-Rab10 or GST, followed by IB with MARCKS antibody. (F) HEK293 cells transfected with GFP-MARCKS were treated with OA (100 nM) or vehicle for 6 h. Cell lysates were subjected to pull-down with glutathione-sepharose beads coupled to GST-Rab10 or GST. Bound proteins were subjected to IB with the indicated antibodies. (G) Rat fibroblasts were treated with DMSO, DES (100 nM), or OA (100 nM) for 2 h, followed by fixation (4% PFA) and penetration with 0.2% saponin, and then stained with antibodies against PIP2 (green), MARCKS (blue), and Rab10 (red). Scale bar, 15 μm. (H) Cell lysates from HEK293 cells transfected with various MARCKS constructs tagged with GFP at the C-terminus were subjected to pull-down with glutathione-sepharose beads coupled to GST-Rab10 (5 μg), followed by IB with GFP or GST antibody. (I) Schematic structure of MARCKS full-length protein or domains. ED, effector domain. (J) Cell lysates from HEK293 cells co-transfected with HA-Rab10 and YFP-tagged MARCKS domains were subjected to IP with anti-HA antibody, and then IB with anti-HA or GFP antibody. (K) GST-Rab10 pull-down of MARCKS domains expressed in HEK293 cells. (I, M) GST-Rab10 immobilized on glutathione-sepharose beads were incubated with purified His6-MARCKS or His6-D3. Bound proteins were subject to IB with the indicated antibodies. (N) Purified GST-Rab10 (2 μg) preloaded with GTPγS or GDP was used to pull down His6-D3. (O) Purified GST-MARCKS was used to pull down His6-Rab10 preloaded with GTPγS or GDP. (P, Q) Lysates of HEK293 cells transfected with the indicated constructs were subject to IP and IB with the indicated antibodies. (R) GST-D3 pull-down of Rab10 (WT or Q68L form) expressed in HEK293 cells.