Figure EV4. BACE1 cleavage does not affect the function of SEZ6 as GluK2/3 regulator.

• A
  WT neurons were treated with the BACE inhibitor C3 or DMSO as control and surface biotinylation with Sulfo‐NHS‐Biotin was performed. BACE1 inhibition did not only prevent SEZ6ecto formation (Pigoni et al, 2016), but also increased full‐length SEZ6 levels at the cell surface compared to the control condition. Even though SEZ6 full length accumulated on the cell surface of BACE‐inhibitor‐treated neurons, no change in GluK2/3 amounts at the cell surface was detected.
• B
  Membrane fraction of WT and BACE1KO brains was digested with EndoH. As expected, SEZ6 full length accumulates in the membrane fraction of BACE1KO brains. No change in GluK2/3 glycosylation or total amount was detected. Color coding for asterisks is the same as in Fig 4A.
• C
  WT and SEZ6KO neurons were treated with the BACE1 inhibitor C3 or DMSO as control and total lysates were analyzed after EndoH digestion. Even though SEZ6 full length accumulates upon BACE inhibition, and GluK2/3 presents immature glycosylation in the SEZ6KO neurons, no change of GluK2/3 glycosylation or total amounts was seen when WT neurons were treated with C3. This indicates that both surface localization (A) and glycosylation (B and C) of GluK2/3 are likely to have reached their maximum in WT cells and cannot be enhanced by increased SEZ6 levels, as induced here through BACE1 inhibition. Color coding for asterisks is the same as in Fig 4A.

Source data are available online for this figure.