Fig 1: AnkR interacting proteins.(A) Immunostaining of mouse cortex using antibodies against AnkR (red) and ß1 spectrin (green). Scalebar, 20 µm. (B) Immunoblot of ß1 spectrin, AnkR, and IgG immunoprecipitation reactions using antibodies against AnkR and ß1 spectrin. IP, immunoprecipitation; IB, immunoblot. (C) Immunoblot of control and AnkR-deficient mouse brains using antibodies against ß1 spectrin, AnkR, and neurofilament-M (NFM). Quantification of the ß1 spectrin immunoblots normalized to NFM. Error bars indicate mean ± SEM. N=6. (D) Top AnkR-interacting candidates. Circle size corresponds to the mean PSM from IP mass spectrometry. Concentric circles radiating from Ank1 correspond to % reduction in PSMs from AnkR knockout mouse compared to control. Identified proteins are organized according to their putative functions. (E–H) Immunostaining of mouse cortex using antibodies against AnkR (red) and TnR (green, E), Bcan (green, F), Acan (green, G), and Vcan (green, H). Scalebars, 20 µm. (I) Immunostaining of mouse cortex and hippocampus using antibodies against AnkR (red) and fluorescent-labeled WFA (green). Scalebars, 250 µm (top) and 20 µm (bottom). (J) Immunostaining of control (left) and AnkR-deficient mouse brain (right) using antibodies against AnkR (red) and NrCAM (green). Scalebars, 50 µm. Normalized fluorescence intensity of perisomatic and axon initial segment (AIS) NrCAM in Ank1F/F (blue) and Ank1F/F;Nes-Cre (gray). Error bars indicate mean ± SEM. N=3. (K) Immunoblot of NrCAM, AnkR, and IgG immunoprecipitation reactions using antibodies against AnkR (top) and NrCAM (bottom). (L) Immunoblot of PlexinA4, AnkR, and IgG immunoprecipitation reactions using antibodies against AnkR (top). Immunoblot of Nrp1, AnkR, and IgG immunoprecipitation reactions using antibodies against AnkR (bottom).Figure 2—source data 1.Source data realted to Figure 2 .