Figure 1. Prosaposin is required for the... | Rockefeller University Press

Figure 1.

$Prosaposin is required for the efficient ER exit of progranulin.(A) Confocal images showing progranulin subcellular localization along with LAMP1-labeled late endosomes/lysosomes in control and prosaposin KD of HeLa cells. Scale bar, 10 µm; inset, 3.38 µm wide. (B) Progranulin localization along with the ER marker, calnexin, in control and prosaposin KD cells. Scale bar, 10 µm; inset, 3.38 µm wide. (C) Image quantification shows the Manders colocalization coefficient of progranulin with LAMP1 in control and prosaposin KD. Data were collected from 20 cells. Error bars show mean ± SEM; unpaired t test; ****, P < 0.0001. (D) Quantification of progranulin ER accumulation (calnexin colocalization) in control and prosaposin KD. Data were collected from 20 cells. Error bars show mean ± SEM; unpaired t test; ****, P < 0.0001. (E) Immunoblot of cell lysates from control siRNA and prosaposin siRNA transfections without (−) or with (+) Endo H enzyme treatment. S, Endo H sensitive; R, Endo H resistant. (F) Quantification of total progranulin protein levels. Total progranulin was calculated by adding Endo H–resistant fraction and Endo H–sensitive fraction. *, P < 0.05. (G and H) Ratios of the Endo H–sensitive (ER-localized) form of progranulin and the Endo H–resistant form of progranulin to total progranulin. (n = 3; mean ± SEM; unpaired t test; **, P < 0.01; *, P < 0.05).$

Prosaposin is required for the efficient ER exit of progranulin. (A) Confocal images showing progranulin subcellular localization along with LAMP1-labeled late endosomes/lysosomes in control and prosaposin KD of HeLa cells. Scale bar, 10 µm; inset, 3.38 µm wide. (B) Progranulin localization along with the ER marker, calnexin, in control and prosaposin KD cells. Scale bar, 10 µm; inset, 3.38 µm wide. (C) Image quantification shows the Manders colocalization coefficient of progranulin with LAMP1 in control and prosaposin KD. Data were collected from 20 cells. Error bars show mean ± SEM; unpaired t test; ****, P < 0.0001. (D) Quantification of progranulin ER accumulation (calnexin colocalization) in control and prosaposin KD. Data were collected from 20 cells. Error bars show mean ± SEM; unpaired t test; ****, P < 0.0001. (E) Immunoblot of cell lysates from control siRNA and prosaposin siRNA transfections without (−) or with (+) Endo H enzyme treatment. S, Endo H sensitive; R, Endo H resistant. (F) Quantification of total progranulin protein levels. Total progranulin was calculated by adding Endo H–resistant fraction and Endo H–sensitive fraction. *, P < 0.05. (G and H) Ratios of the Endo H–sensitive (ER-localized) form of progranulin and the Endo H–resistant form of progranulin to total progranulin. (n = 3; mean ± SEM; unpaired t test; **, P < 0.01; *, P < 0.05).