Fig. 6: Features of CID-CLC interactions.

a Phosphorylation-mimicking mutations in ClC-3 CBS1 weaken inhibition by T9A and T9B. Left, immunocytochemistry; right, live cell imaging. b Phosphorylation-mimicking mutations in T9B ¹⁹⁰Ser weaken inhibition of vacuolization. Immunocytochemistry (left) and live cell imaging (right). c T9 co-expression change ClC−5 localization from ER-like (top) to punctate endosome-like (lower panels), shown by immunocytochemistry. d T9A^ΔCID or T9B^ΔCID co-expression with ClC-5 induces vacuolization. e Neither T9A nor T9A^ΔCID changes ER-like pattern of ClC-4 expression, nor induces vacuolization. f Co-expressing transport-deficient ClC-3^td with ClC-4 induces vacuolization that is suppressed by T9A but not T9A^ΔCID. g Co-expressing WT ClC-3 and ClC-4 leads to T9-suppressible vacuolization. h Co-expressing WT ClC-3 with disease mutant ClC-4^R360S leads to T9-resistant vacuolization. e–h phase contrast (left) of live cell images for Lysosensor® (right). Representative images in (b–d) and (f, g) obtained from technical duplicates and n≥3 biological replicates. Split optical channels depicted from the zoomed areas in (a–d). All scale bars, 10 µm.