Figure 3. IFT20 interacts with the BLOC-1 subunit pallidin and the exocyst subunits Exo70 and Sec8. (A) Flag-GFP (negative IFT20 binding control), Flag-IFT54 (positive IFT20 binding control), Flag-KXD1, Flag-Pallidin, and Flag-Exo70 were expressed in mouse IMCD3 cells. The cells were lysed, immunoprecipitated, with Flag antibody and analyzed by immunoblotting. (Aa) The left group is the starting material before immunoprecipitation (10% input), and (Ab) the right group is the precipitated material (IP). Flag-tagged proteins are marked with arrows on the Flag Western blot. Antibodies used for the Western blots are listed on the right side. (B) Flag-GFP (negative binding control), Flag-IFT25 (positive binding control for IFT complex B protein IFT88), and Flag-IFT20 were expressed in mouse IMCD3 cells. The cells were lysed, immunoprecipitated, with Flag antibody and analyzed by immunoblotting. The left group is the starting material before immunoprecipitation (10% input), and the right group is the precipitated material. Proteins are marked with arrows. Antibodies used for the Western blots are listed on the right side. (C) Selected images of IMCD3 cells expressing the Flag-fusion proteins that are listed horizontally at the top of the figure. Flag-fusion proteins were detected with Flag antibody staining (green), endogenous IFT20 antibody staining (red), and nuclei detected with DAPI (blue). (Ca) IFT20 is localized at the Golgi membranes in IMCD3 control cells (arrows). (Cb) Increased pixel intensity of IFT20 in the cytoplasm (arrows) and complete loss of IFT20 from the perinuclear Golgi pool in Flag-IFT54-expressing cells. (Cc) Flag-KXD1-expressing cells show IFT20 localization at the Golgi membranes (arrows) and no IFT20 displacement into the cytoplasm. (Cd) Compacted Golgi membranes (asterisks) and increased pixel intensity of IFT20 in the cytoplasm (arrows) and partial displacement from the Golgi membranes in Flag-Pallidin-expressing cells. (Ce) Up-regulated filopodia (asterisks) formation and increased IFT20 pixel intensity (arrows), with partial IFT20 displacement from Golgi membranes in cells expressing Flag-Exo70. Bar, 10 µm. (D) Selected images of IMCD3 cells expressing the Flag-fusion proteins that are listed horizontally at the top of the figure. Flag-fusion proteins were detected with Flag antibody staining (green), endogenous GMAP210 antibody staining (red), and nuclei detected with DAPI (blue). (Da) GMAP210 is localized at the Golgi membranes in IMCD3 control cells, (Db) Flag-IFT54-expressing cells, and (Dc) Flag-KXD1-expressing cells (arrows). (Dd) Compacted Golgi membranes (asterisks) and GMAP210 are localized at the Golgi membranes (arrows) in Flag-Pallidin-expressing cells. (De) Up-regulated filopodia (asterisks) formation and GMAP210 are localized at the Golgi membranes (arrows) in cells expressing Flag-Exo70. Bar, 10 µm.
Figure 3.

IFT20 interacts with the BLOC-1 subunit pallidin and the exocyst subunits Exo70 and Sec8. (A) Flag-GFP (negative IFT20 binding control), Flag-IFT54 (positive IFT20 binding control), Flag-KXD1, Flag-Pallidin, and Flag-Exo70 were expressed in mouse IMCD3 cells. The cells were lysed, immunoprecipitated, with Flag antibody and analyzed by immunoblotting. (Aa) The left group is the starting material before immunoprecipitation (10% input), and (Ab) the right group is the precipitated material (IP). Flag-tagged proteins are marked with arrows on the Flag Western blot. Antibodies used for the Western blots are listed on the right side. (B) Flag-GFP (negative binding control), Flag-IFT25 (positive binding control for IFT complex B protein IFT88), and Flag-IFT20 were expressed in mouse IMCD3 cells. The cells were lysed, immunoprecipitated, with Flag antibody and analyzed by immunoblotting. The left group is the starting material before immunoprecipitation (10% input), and the right group is the precipitated material. Proteins are marked with arrows. Antibodies used for the Western blots are listed on the right side. (C) Selected images of IMCD3 cells expressing the Flag-fusion proteins that are listed horizontally at the top of the figure. Flag-fusion proteins were detected with Flag antibody staining (green), endogenous IFT20 antibody staining (red), and nuclei detected with DAPI (blue). (Ca) IFT20 is localized at the Golgi membranes in IMCD3 control cells (arrows). (Cb) Increased pixel intensity of IFT20 in the cytoplasm (arrows) and complete loss of IFT20 from the perinuclear Golgi pool in Flag-IFT54-expressing cells. (Cc) Flag-KXD1-expressing cells show IFT20 localization at the Golgi membranes (arrows) and no IFT20 displacement into the cytoplasm. (Cd) Compacted Golgi membranes (asterisks) and increased pixel intensity of IFT20 in the cytoplasm (arrows) and partial displacement from the Golgi membranes in Flag-Pallidin-expressing cells. (Ce) Up-regulated filopodia (asterisks) formation and increased IFT20 pixel intensity (arrows), with partial IFT20 displacement from Golgi membranes in cells expressing Flag-Exo70. Bar, 10 µm. (D) Selected images of IMCD3 cells expressing the Flag-fusion proteins that are listed horizontally at the top of the figure. Flag-fusion proteins were detected with Flag antibody staining (green), endogenous GMAP210 antibody staining (red), and nuclei detected with DAPI (blue). (Da) GMAP210 is localized at the Golgi membranes in IMCD3 control cells, (Db) Flag-IFT54-expressing cells, and (Dc) Flag-KXD1-expressing cells (arrows). (Dd) Compacted Golgi membranes (asterisks) and GMAP210 are localized at the Golgi membranes (arrows) in Flag-Pallidin-expressing cells. (De) Up-regulated filopodia (asterisks) formation and GMAP210 are localized at the Golgi membranes (arrows) in cells expressing Flag-Exo70. Bar, 10 µm.

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