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. 2016 Mar 25;73(18):3599–3621. doi: 10.1007/s00018-016-2183-4

Fig. 2.

Fig. 2

Mint1 competes with liprin-α for CASK interaction. a Domain structure of CASK showing the regions included in the GST-CaMK and GST-CASK constructs. CASK antibody used in immunoprecipitation was raised against L27 motifs. b CASK antibody was used to immunoprecipitate CASK and binding partners from mouse brain lysate. A cartoon diagram representing the immunoprecipitation experiment is shown below the panel. Starting material is shown on the left of the arrow and predominant species pulled down is shown on the right of the arrow. CASK, red; Mint1, purple. c HEK293 cells were transiently transfected with cDNAs for GFP-CASK, liprin-α3, or FLAG-tagged Mint1, as indicated. GFP-CASK was precipitated from solubilized cells using GFP-trap™ beads, along with any other interacting proteins, and blots were performed with indicated antibodies. Cartoon diagrams representing the pulldown experiment is shown to the right of the panel. Starting material is shown on the left of the arrow and predominant species pulled down is shown on the right of the arrow. CASK, red; Mint1, purple; liprin-α, green. d Quantification of band intensity levels from c, showing differences in the percent of input liprin brought down by CASK in the presence or absence of FLAG-Mint1 (mean and SEM; n = 3; asterisk indicates significance of p < 0.05). e Glutathione beads bound with either GST-CaMK or GST-CASK (full-length) were used to pull down CASK binding partners from mouse brain lysate. Left panel Ponceau S-stained gel demonstrating amounts of sample loaded on gel. Right panel blots for indicated proteins (synaptophysin, negative control; neurexin1β, positive control). Cartoon diagrams representing the pulldown experiment is shown to the right. Pulldown strategy is indicated by entity over the arrow (glutathione beads with attached CAMK domain or full-length CASK). Starting material is shown on the left of the arrow and predominant species pulled down is shown on the right of the arrow. CASK, both full-length and CAMK domain, red; Mint1, purple; liprin-α, green; neurexin, blue. f Quantification of band intensity levels from e shows differences in the percent of input liprin and neurexin brought down by either full-length CASK or the CAMK domain of CASK (mean and SEM; n = 3; asterisk indicates significance of p < 0.05)