Fig 3

(A) HeLa cells were transfected with mCherry or mCherry-TBC1D23 FL (“FL”), ΔRhod2 (deleting aa331–460), or C399S/R405A (399/405) and then fixed and labeled with anti-ZFPL1 (Golgi marker, green) antibody. Scale bar: 10 μm. (B) Quantitation of mCherry colocalization with ZFPL1 in cells as treated in (A). Each dot represents Pearson’s correlation coefficients from one cell. P values were calculated using one-way ANOVA, post hoc Tukey’s test. ***P < 0.001. Experiments were triplicated, and the numerical data are included in S1 Data. (C) TBC1D23-KO HeLa cells were transfected with mCherry or FL, ΔRhod2, or 399/405 and then fixed and labeled with anti-TGN46 (green) and GM130 (Golgi marker, white) antibodies. TGN46 is recycled between endosomes and Golgi in a TBC1D23-dependent manner. Scale bar: 10 μm. (D) Quantitation of Golgi-localized TGN46 over its total amount in cells treated as in (C). Each dot represents results from one cell. P values were calculated using one-way ANOVA, post hoc Tukey’s test. ***P < 0.001. Experiments were triplicated, and the numerical data are included in S1 Data. (E) Immunoblot of whole-cell extracts showing the total protein level of CI-MPR and TGN46 in cells treated in (C). aa, amino acid; CI-MPR, cation-independent mannose-6-phosphate receptor; FL, full-length; GAPDH, glyceraldehyde 3-phosophate dehydrogenase; KO, knockout; ns, not significant; TGN, trans-Golgi network; ZFPL1, zinc finger protein like 1.