Fig 5

(A) GST pull-down analyses of HEK 293T cells transfected with vectors expressing GST, GST-TBC1D23-FL (“FL”), GST-TBC1D23-ΔTBC (deleting aa1–330), or GST-TBC1D23-ΔRhod1 (deleting aa331–513). Cell lysates were precipitated with glutathione-Sepharose beads and probed with anti-GST, golgin-97, or GAPDH (control) antibodies. (B) GST pull-down analyses of HEK 293T cells transfected with vectors expressing GST, GST-TBC1D23-FL (“FL”), TBC1D23-L278A/Y281A/Y282A (“278/281/282”), TBC1D23-I236A/I237A/V239A (“236/237/239”), or TBC1D23-E425K/Y426A (“425/426”). Cell lysates were precipitated with glutathione-Sepharose beads and probed with anti-GST, golgin-97, or GAPDH (control) antibodies. (C) HeLa cells were transfected with mCherry, or mCherry-TBC1D23 FL (“FL”), TBC1D23-L278A/Y281A/Y282A (“278/281/282”), TBC1D23-I236A/I237A/V239A (“236/237/239”), or TBC1D23-E425K/Y426A (“425/426”) and then fixed and labeled with anti-ZFPL1 (blue) antibody. Scale bar: 10 μm. (D) Quantitation of mCherry colocalization with ZFPL1 (Golgi marker) in cells as treated in (C). 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. (E) Immunoblot of whole-cell extracts of parental HeLa Cells (control), TBC1D23 KO cells (“KO”), TBC1D23 KO cells transfected with vectors expressing mCherry, mCherry-TBC1D23-FL (“FL”), TBC1D23-L278A/Y281A/Y282A (“278/281/282”), TBC1D23-I236A/I237A/V239A (“236/237/239”), or TBC1D23-E425K/Y426A (“425/426”). Cell lysates were probed with anti-cherry, TBC1D23, TGN46, or actin (control) antibodies. (F) Confocal immunofluorescence of TBC1D23 KO HeLa cells transfected with vectors expressing mCherry, mCherry-TBC1D23-FL (“FL”), TBC1D23-L278A/Y281A/Y282A (“278/281/282”), TBC1D23-I236A/I237A/V239A (“236/237/239”), or TBC1D23-E425K/Y426A (“425/426”). The cells were fixed and labeled with anti-TGN46 (green) and GM130 (white) antibodies. Scale bar: 10 μm. (G) Quantitation of Golgi-localized TGN46 over its total amount in cells treated as in (F). 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. aa, amino acid; FL, full-length; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione S-transferase; HEK, human embryonic kidney; KO, knockout; TBC, Tre2-Bub2-Cdc16; TGN, trans-Golgi network; ZFPL1, zinc finger protein like 1.