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Fig. 1 | BMC Biology

Fig. 1

From: Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition

Fig. 1

p35 interacts with JIP1. A Identification of JIP1 as p35-binding protein. Four peptides were identified and matched to mouse JIP1 by tandem mass spectrometry. B One microgram of bacterially expressed GST or GST fused-JIP1 was incubated with 2 μg His-p35 at 4°C for 2 h. The GST-tagged proteins were retrieved with GSH beads. The proteins were subjected to SDS-PAGE and detected using anti-p35 antibody by western blot. Amido black was used to visualize protein levels. C Endogenous p35 interacts with JIP1 in brain lysate. One microgram of IgG as control or JIP1 antibody was incubated with 500 μg of mouse brain lysate. Antibodies were pulled down using 50 μl goat-anti-rabbit IgG beads and the coupled proteins were subjected to SDS-PAGE followed by western blotting with anti-p35 antibody. D Confirmation of the specific interaction between p35 and JIP1 using p35 KO cortical neurons. Control IgG or anti-p35 was incubated with lysates from p35 WT or KO cortical neurons. The IPed proteins were immunoblotted with anti-JIP1 antibody. E Two-microgram control IgG or anti-p35 was incubated with lysate from JIP1 WT and KO neurons and the IPed proteins were immunoblotted with anti-JIP1 antibody. Images are representative of at least three independent experiments

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