Figure S1. In vitro ER budding in SICs... | Rockefeller University Press

Figure S1.

$In vitro ER budding in SICs derived from murine primary neurons and rat primary glial cells and isolation of secretory pathway compartments.(A) SDS-PAGE and Western blot analysis of in vitro generated COPII vesicles from murine primary cortical neurons (7 d in vitro) and rat glial SICs: 4.2% of SICs (input) and 100% of COPII-coated vesicles were analyzed with the indicated antibodies. Reactions without nucleotides were used as a negative control. COPII budding was specifically inhibited by the GTP-restricted mutant Sar1b H79G. A representative Western blot shows COPII-dependent budding of immature NCT, FL PSEN1, and APH1A in both murine cortical neurons and rat glia (filled arrowheads: immature NCT; open arrowheads: FL PSEN1; asterisk: aspecific band). (B–D) Enrichment of the ER. Fractionation of MEF WT and MEF PSEN-dKO using a continuous 10–24% Nycodenz gradient followed by Western blotting (equal fraction volume per lane) for (B) ribophorin, calnexin, KDEL: ER; ERGIC53/p58: ERGIC; GM130: cis-Golgi; syntaxin 6: trans-Golgi Network (TGN); TfR: PM and recycling endosomes; EEA1, Rab5, Hrs: early endosomes (EE); Rab7: late endosomes (LE); (C) γ-secretase components; and (D) γ-secretase activity (after a cell-free γ-secretase assay). ER proteins are most strongly enriched in fractions 10 and 11, which were pooled as “enriched ER” for BN-PAGE. In WT cells, mature γ-secretase complex members (indicated by mature glycosylated NCT and PSEN1-NTF and –CTF) and de novo Aβ production, representing γ-secretase activity, comigrate only in fractions enriched for endosomal markers. In PSEN-deficient cells, immature NCT and PEN-2 are largely located to ER fractions. (E) γ-Secretase complex members are enriched in the ERGIC. Purification of the ERGIC combining Percoll gradient and discontinuous Nycodenz gradient centrifugation (see Materials and methods for details). The second step was needed to further separate the ERGIC from the trans-Golgi network (TGN), as shown by the comparative Western blot analysis of TH and PNS with the 18.5–27% interface of the Percoll gradient (I1) and final enriched ERGIC (equal amount of protein per lane). The ERGIC fraction is also devoid of the cis-Golgi marker GM130 but still contaminated with ER (calnexin). γ-Secretase complex members NCT, APH1, and PEN-2 coenrich with ERGIC-53/p58 with a stronger relative enrichment of immature NCT as expected from early biosynthetic compartments. (F) Magnetic isolation of PM from WT and PSEN-dKO MEFs using SPIONs. A representative Western blot showing PNS (postnuclear supernatant), UB (unbound fraction), and B (bound fraction) with quantification (n = 4 independent isolations) show strong enrichment for the PM marker, Na+K+-ATPase, along with PSEN1 and mature NCT, whereas the ER marker, ribophorin, does not enrich and the late endosomal Rab7 enriches only moderately, as described (Thimiri Govinda Raj et al., 2011).$

In vitro ER budding in SICs derived from murine primary neurons and rat primary glial cells and isolation of secretory pathway compartments. (A) SDS-PAGE and Western blot analysis of in vitro generated COPII vesicles from murine primary cortical neurons (7 d in vitro) and rat glial SICs: 4.2% of SICs (input) and 100% of COPII-coated vesicles were analyzed with the indicated antibodies. Reactions without nucleotides were used as a negative control. COPII budding was specifically inhibited by the GTP-restricted mutant Sar1b H79G. A representative Western blot shows COPII-dependent budding of immature NCT, FL PSEN1, and APH1A in both murine cortical neurons and rat glia (filled arrowheads: immature NCT; open arrowheads: FL PSEN1; asterisk: aspecific band). (B–D) Enrichment of the ER. Fractionation of MEF WT and MEF PSEN-dKO using a continuous 10–24% Nycodenz gradient followed by Western blotting (equal fraction volume per lane) for (B) ribophorin, calnexin, KDEL: ER; ERGIC53/p58: ERGIC; GM130: cis-Golgi; syntaxin 6: trans-Golgi Network (TGN); TfR: PM and recycling endosomes; EEA1, Rab5, Hrs: early endosomes (EE); Rab7: late endosomes (LE); (C) γ-secretase components; and (D) γ-secretase activity (after a cell-free γ-secretase assay). ER proteins are most strongly enriched in fractions 10 and 11, which were pooled as “enriched ER” for BN-PAGE. In WT cells, mature γ-secretase complex members (indicated by mature glycosylated NCT and PSEN1-NTF and –CTF) and de novo Aβ production, representing γ-secretase activity, comigrate only in fractions enriched for endosomal markers. In PSEN-deficient cells, immature NCT and PEN-2 are largely located to ER fractions. (E) γ-Secretase complex members are enriched in the ERGIC. Purification of the ERGIC combining Percoll gradient and discontinuous Nycodenz gradient centrifugation (see Materials and methods for details). The second step was needed to further separate the ERGIC from the trans-Golgi network (TGN), as shown by the comparative Western blot analysis of TH and PNS with the 18.5–27% interface of the Percoll gradient (I1) and final enriched ERGIC (equal amount of protein per lane). The ERGIC fraction is also devoid of the cis-Golgi marker GM130 but still contaminated with ER (calnexin). γ-Secretase complex members NCT, APH1, and PEN-2 coenrich with ERGIC-53/p58 with a stronger relative enrichment of immature NCT as expected from early biosynthetic compartments. (F) Magnetic isolation of PM from WT and PSEN-dKO MEFs using SPIONs. A representative Western blot showing PNS (postnuclear supernatant), UB (unbound fraction), and B (bound fraction) with quantification (n = 4 independent isolations) show strong enrichment for the PM marker, Na⁺K⁺-ATPase, along with PSEN1 and mature NCT, whereas the ER marker, ribophorin, does not enrich and the late endosomal Rab7 enriches only moderately, as described (Thimiri Govinda Raj et al., 2011).