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. 2017 Aug 2;595(17):5913–5930. doi: 10.1113/JP274790

Figure 4. KAR subunits are substrates for HNK‐1 conjugation.

Figure 4

A, immunoprecipitation of the HNK‐1 epitope with KAR subunits. Recombinant KAR subunits were co‐expressed in HEK293T/17 cells with or without GlcAT‐P and HNK‐1ST, as denoted by the table of transfection combinations. KAR subunits were immunoprecipitated from cell lysates with an antibody against myc or against the GluK2 and GluK3 subunits. HNK‐1 was detected by immunoblotting for the HNK‐1 epitope; enrichment of KAR subunits was confirmed by immunoblotting for myc or GluK2/3. B, western blot showing that KAR subunits in the hippocampus contain the HNK‐1 epitope. KARs were precipitated from hippocampal homogenates using an antibody against GluK2 and GluK3. HNK‐1 is detected by immunoblotting in immunoprecipitates from wild‐type mice but not from mice lacking all five KAR subunits (KAR−/−). Input lanes (10 μg) show that HNK‐1 is present on other proteins in KAR−/− tissue. Membranes were stripped and re‐probed for GluK2/3 to confirm KAR subunit enrichment. C, HNK‐1 was detected on hippocampal KAR subunits following immunoprecipitation with anti‐GluK2/3 from wild‐type mice but not from mice lacking the HNK‐1ST (HNK−/−). Membranes were stripped and re‐probed for GluK2/3 to confirm KAR subunit enrichment.