Surf4 binds prosaposin–progranulin complex in the ER and facilitates ER exit. (A) Immunofluorescence images of endogenous progranulin and calnexin (ER marker) in control, Surf4 KO, and Surf4 KO cells expressing GFP-Surf4. Scale bar, 10 µm; inset, 3.38 µm wide. (B and C) Immunofluorescence images of endogenous progranulin and prosaposin along with LAMP1 (late endosomes and lysosomes) in control, Surf4 KO, and Surf4 KO cells expressing GFP-Surf4. Scale bar, 10 µm; inset, 3.38 µm wide. (D) Quantification of progranulin accumulation in ER (calnexin colocalization) in Surf4 KO cells. Data were collected from n = 25–32 cells. Error bars show mean ± SEM. Kruskal–Wallis test with Dunn’s multiple comparisons test; ***, P < 0.001; ****, P < 0.0001. (E and F) Quantification of Manders colocalization coefficients for progranulin and prosaposin with LAMP1 in control and Surf4 KO cells. Data were collected from three independent experiments with n = 20 cells quantified per experiment. Error bars show mean ± SEM. One-way ANOVA with Sidak’s multiple comparisons test; ****, P < 0.0001. (G) Anti-GFP-Surf4 IPs support an interaction with prosaposin and show that prosaposin enhances the abundance of endogenous progranulin in the complex with Surf4. Similar results were observed in three independent experiments.