egfp abi1  (Addgene inc)

Journal: Biochemical and biophysical research communications

Article Title: CaMKIIα Phosphorylation of Shank3 Modulates ABI1-Shank3 Interaction

doi: 10.1016/j.bbrc.2020.01.089

Figure Lengend Snippet: A, B. Lysates of HEK293T cells expressing HA-ABI1 (A) or HA-CaV1.3-CTD (B) and either empty vector, GFP-Shank3-WT, GFP-Shank3-S685A, or GFP-Shank3-S685D were immunoprecipitated (IP) using a GFP antibody. Input samples and IP complexes were resolved by SDS-PAGE and immunoblotted for GFP-Shank3 and HA-ABI1 (A) or HA-CaV1.3-CTD (B). HA/GFP signals for each IP lane were calculated and normalized to GFP-Shank3-WT. Both GFP-Shank3–685A and GFP-Shank3–685D significantly reduce HA-ABI1 co-immunoprecipitation (GFP-Shank3-S685A: 37±11% reduced compared to WT, ** p < 0.01, GFP-Shank3-S685D: 32±8% reduced compared to WT, *** p < 0.001, one sample Student’s t-test with equal variance compared to theoretical mean of 100). Mutation of S685 had no effect on HA-CaV1.3-CTD co-immunoprecipitation (one sample Student’s t-test with equal variance compared to theoretical mean of 100). The immunoblots shown are representative of 4–5 biological replicates that were quantified. Error bars, mean ± SEM.

Article Snippet: The eGFP-ABI1 vector was purchased from Addgene (#74905), and DNA encoding full-length ABI1 was PCR amplified and inserted into the pCGNh vector, a gift from Dr. Winship Herr (Université de Lausanne), between Xba1 and BamHI restriction sites to generate N-terminal HA-tagged ABI1.

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Mutagenesis, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: CaMKIIα Phosphorylation of Shank3 Modulates ABI1-Shank3 Interaction

doi: 10.1016/j.bbrc.2020.01.089

Figure Lengend Snippet: Left, GST, GST-Shank3 WT, or GST-Shank3-S685A were pre-phosphorylated with CaMKIIα and incubated with HEK293T cell lysate expressing HA-ABI1. Control protein samples were pre-incubated in the absence of CaMKIIα. Isolated GST complexes were then analyzed by immunoblotting as indicated (see Methods). Immunoblots are representative of 5 independent experiments. Right, HA/GST signals from each pulldown lane were normalized to GST-Shank3-WT/no CaMKII condition. Incubation with CaMKIIα significantly increases HA-ABI1 binding to GST-Shank3-WT (148±14% increased relative to no CaMKII condition, * p < 0.05, one sample Student’s t-test with equal variance compared to theoretical mean of 100), which is not observed with GST-Shank3-S685A (unpaired Student’s t-test with equal variance). Error bars, mean ± SEM.

Article Snippet: The eGFP-ABI1 vector was purchased from Addgene (#74905), and DNA encoding full-length ABI1 was PCR amplified and inserted into the pCGNh vector, a gift from Dr. Winship Herr (Université de Lausanne), between Xba1 and BamHI restriction sites to generate N-terminal HA-tagged ABI1.

Techniques: Incubation, Expressing, Control, Isolation, Western Blot, Binding Assay