Figure 2. Ser¹¹⁸¹Ala substitution and RSLESD deletion disrupt binding of L1 to 14-3-3ζ.

A. Schematic of recombinant L1ICD constructs. The full-length L1ICD construct contains the RSLESD sequence, a potential 14-3-3 binding motif. L1ICD S1181A has a single amino acid substitution (S1181A) of a serine residue in this motif. Ser1181 can be phosphorylated by CKII. The RSLESD sequence, a potential 14-3-3-binding motif, is deleted in L1ICD ΔRSLESD. Wild-type and mutated L1ICD constructs were recombinantly expressed as 6xHis-tagged proteins, purified from bacterial lysates and used in pull-down experiments. B. Purified mutated L1ICD constructs were analyzed by Western blot using the 74-5H7 anti-L1 antibody. C. Upper panel: 6xHis-tagged proteins, purified from bacterial lysates, were subjected to GST-14-3-3ζ pull-down assays after treatment with CKII. GST was used as a control. Pull-down eluates were analyzed by Western blot (WB) with the 74-5H7 anti-L1 antibody. Lower panel, GST and GST-14-3-3ζ were detected in pull-down eluates by Western blot (WB) with an anti-GST antibody, confirming that comparable amounts of GST or GST-14-3-3ζ were used in each pull-down.