Journal: The Journal of Neuroscience

Article Title: Trip6 Promotes Dendritic Morphogenesis through Dephosphorylated GRIP1-Dependent Myosin VI and F-Actin Organization

doi: 10.1523/JNEUROSCI.2125-14.2015

Figure Lengend Snippet: GRIP1956T is phosphorylated by AKT1. A, GFP-LR2+PDZ7 (GRIP1 fragment) was immunoprecipitated (IP) with anti-GFP antibody and separated by SDS-PAGE followed by mass spectrometry analysis. Threonine at the 956 position (956T) was identified to be phosphorylated (red box). Co. St., Coomassie blue staining. B, Alignment of the amino acid sequence containing GRIP1855T (negative control) and GRIP1956T among different eukaryotes; 956T is emphasized in bold and marked by an asterisk; the predicted AKT1-phosphorylated motif is indicated by the arrow. C, Overexpressed GFP-LR2+PDZ7 (GRIP1 fragment) was immunoprecipitated from HEK293T cells with anti-GFP antibody and treated with lambda phosphatase (λ PPase) or calf intestine alkaline phosphatase (CIAP) followed by immunoblotting (IB) with anti-GRIP1956T phosphorylation-specific antibody (pGRIP1). D, WT and phosphodead mutants T855A, T956A, and both T855A and T956A (T855A+T956A) of GFP-LR2+PDZ7 were transfected into HEK293T cells and analyzed by IB with pGRIP1 antibody. E, F, IB of phospho-GRIP1956T (pGRIP1) and total GRIP1 (tGRIP1) in the whole-brain (E) and hippocampus (F) homogenates of mice at different developmental stages. E, Embryonic day; P, Postnatal day; PW, Postnatal week. G, Pull-down of overexpressed GFP-AKT1 by GST-LR2+PDZ7 in HEK293T cell lysates. H, GFP-LR2+PDZ, together with AKT1 shRNA vector (628 or 642), was transfected into HEK293T cells for 72 h followed by IB. I, J, HEK293T cells overexpressing WT (I) or T956A (J) of GFP-LR2+PDZ7 were treated with insulin alone or together with wortmannin following serum starvation for 36 h and then analyzed by IB with pGRIP1 antibody. K, Cultured hippocampal neurons were treated with insulin alone or together with wortmannin following serum starvation at 7 DIV for 36 h and then analyzed by IB with pGRIP1 antibody.

Article Snippet: One aliquot was left untreated, and the other two were washed extensively with their respective buffers before treatment either with 400 U lambda phosphatase (Merck) at 30°C for 30 min or with 400 U of calf intestinal alkaline phosphatase (Promega) at 37°C for 30 min.

Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry, Staining, Sequencing, Negative Control, Western Blot, Transfection, shRNA, Plasmid Preparation, Cell Culture