WDR44 labels a subset of tubular endosomes. (A) Confocal stacks of hTERT-RPE1, 293T, HeLa, U-87 MG, HT 1080, and COS-7 cells incubated with BFA (5 µg/ml, 15 min) and stained with α-WDR44. All cell types showed an increase in endogenous WDR44 tubules (compare with Fig. S3 E). (B) Confocal images of transfected HeLa cells left untreated or after incubation with BFA (5 µg/ml, 15 min) and stained with α-WDR44. While there was no colocalization of endogenous WDR44 tubules with GFP-STX16 or GFP-VAMP3 under resting conditions, colocalization was seen after incubation with BFA. (C) Manders colocalization coefficients for GFP-STX16 and GFP-VAMP3 with endogenous WDR44 structures. n = 10–20 cells. Data are means ± SEM; **, P < 0.01; and ****, P < 0.0001. (D) Confocal images of untransfected HeLa cells incubated with BFA (5 µg/ml) for 5 or 15 min and stained with α-WDR44 and α-ERGIC53, α-GM130, or α-LAMP2. Endogenous WDR44 tubules did not colocalize with any of these proteins. (A, B, and D) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.