Figure 1. Fra is cleaved by γ-secretase.

A) Protein extracts were made from embryos pan-neurally expressing either a full-length Fra receptor with a C-terminal 6× Myc tag (first lane) or an equivalently tagged Fra ICD (second lane). Proteins were resolved by SDS-PAGE and Western blots were performed with anti-Myc antibody. We detect full-length receptor at approximately 200 kDa (FL) and several C-terminal fragments, including species at approximately 50 kDa, 35 kDa, and 25 kDa (ICD A, ICD B, and ICD C; indicated by asterisks).

B) Protein extracts were made from embryos pan-neurally expressing either a full-length Fra receptor with a C-terminal 3× HA tag (first lane) or a Fra receptor missing its cytoplasmic domain (second lane) and HA-tagged proteins were immunoprecipitated from these extracts with anti-HA antibody (third and fourth lanes). Proteins were resolved by SDS-PAGE and Western blots were performed with anti-HA antibody. The 3× HA tag is smaller than the 6× Myc tag and, accordingly, ICD A, ICD B, and ICD C are shifted to smaller sizes of approximately 45 kDa, 30 kDa, and 20 kDa in both total protein extracts and immunoprecipitates (first and third lanes, indicated by asterisks). These species are not detected in extracts or immunoprecipitates from embryos expressing FraΔC (second and fourth lanes). The position of the IgG heavy chain in the lanes that contain immunoprecipitates is indicated.

C) Schematic of strategy used to express UAS-Fra-Myc with elav-Gal4 specifically in psn mutants.

D) Schematic of strategy used to express UAS-Fra-HA with elav-Gal4 specifically in aph-1 mutants.

E) Protein extracts from embryos pan-neurally expressing Fra-Myc in psn¹² mutants were resolved by SDS-PAGE and Western blots were performed with anti-Myc antibody. All three C-terminal fragments (indicated by asterisks) are reduced in abundance relative to full-length receptor in the total lysates (compare first and second lanes) and the two smaller fragments are reduced in abundance in immunoprecipitates (compare third and fourth lanes). ICD A is obscured in immunoprecipitates by the IgG heavy chain.

F) Quantification of Fra ICD fragments in total lysates relative to full-length receptor in psn¹²/+ compared to psn¹²/psn¹². Data were analyzed by Student's t-test. ** indicates p<0.005. Error bars indicate standard deviation. Data are from six independent experiments.

G) Protein extracts from embryos pan-neurally expressing Fra-HA in aph-1^D35 mutants were made and HA-tagged proteins were immunoprecipitated with anti-HA antibody. Proteins were resolved by SDS-PAGE and Western blots were performed with anti-HA antibody. The two smaller fragments (indicated by asterisks) are reduced in abundance in immunoprecipitates. The largest fragment is obscured by the IgG heavy chain.

H) Quantification of Fra ICD fragments relative to full-length receptor in aph-1^D35/+ compared to aph-1^D35/ aph-1^D35. Data were analyzed by Student's t-test. ** indicates p<0.005. Error bars indicate standard deviation. Data are from four independent experiments.