Figure 2. Progranulin interactions with... | Rockefeller University Press

Figure 2.

$Progranulin interactions with prosaposin begin in the ER.(A) Endo H–treated or untreated IP fractions of mCherry-progranulin. (B and C) Quantification of Endo H–sensitive and –resistant forms of progranulin and prosaposin in IP fractions (n = 5 independent experiments; mean ± SEM). (D) RUSH strategy: mCherry-progranulin fused to SBP is retained in the ER via its interaction with KDEL-streptavidin. Addition of biotin disrupts this interaction and causes the synchronous release of SBP-mCherry-progranulin from the ER. (E) Localization of RUSH-progranulin at 0 and 30 min after biotin addition. HeLa cells were cotransfected with RUSH-progranulin and GFP-GalT (Golgi marker) 1 d before imaging. Scale bar, 10 µm. (F) Immunoblot of prosaposin in IP fractions of RUSH-progranulin at 0 and at 30 min after biotin treatment. (G) Quantification of immunoblot shows the ratio of prosaposin to RUSH-progranulin at 0 and 30 min after biotin treatment (n = 3 independent experiments; mean ± SEM).$

Progranulin interactions with prosaposin begin in the ER. (A) Endo H–treated or untreated IP fractions of mCherry-progranulin. (B and C) Quantification of Endo H–sensitive and –resistant forms of progranulin and prosaposin in IP fractions (n = 5 independent experiments; mean ± SEM). (D) RUSH strategy: mCherry-progranulin fused to SBP is retained in the ER via its interaction with KDEL-streptavidin. Addition of biotin disrupts this interaction and causes the synchronous release of SBP-mCherry-progranulin from the ER. (E) Localization of RUSH-progranulin at 0 and 30 min after biotin addition. HeLa cells were cotransfected with RUSH-progranulin and GFP-GalT (Golgi marker) 1 d before imaging. Scale bar, 10 µm. (F) Immunoblot of prosaposin in IP fractions of RUSH-progranulin at 0 and at 30 min after biotin treatment. (G) Quantification of immunoblot shows the ratio of prosaposin to RUSH-progranulin at 0 and 30 min after biotin treatment (n = 3 independent experiments; mean ± SEM).