Fig 1: The loss of SCFD1 affects the interaction of ACBD3 with giantin.(A) GFP alone and GFP-ACBD3 were overexpressed in either WT HeLa, SCFD1-KO or a double-KO for golgin-45 and giantin. Immunoprecipitation experiments were performed, and immunoprecipitates were blotted for endogenous SCFD1, giantin, and golgin-45. The experiment was performed two times independently. (B) Quantification of giantin levels in immunoprecipitation experiment, expressed in relation to giantin levels from control condition using GFP-ACBD3 WT. An average was taken from two experiments. Error bars = SEM. (C) Same as (B) but for SCFD1. (D) Same as (B and C), but for golgin-45 levels in lysates. (E) Validation of guide RNA KO efficiency by qRT-PCR. Data from all conditions were internally normalized to GAPDH and TBP expression and are represented as fold change of control HeLa Cas9 cells. Error bar = SD of three technical repeats. ***P ≤ 0.001.
Fig 2: ACBD3 interacts and colocalizes with SCFD1.(A) SCFD1 colocalizes with ACBD3 at the Golgi. Widefield imaging of HeLa cells transfected with SCFD1-HaloTag and stained for endogenous ACBD3. Three biological repeats, nucleus stain = DAPI. Scale bar: 10 μm. (B) ACBD3 interacts with SCFD1. GFP alone or GFP-ACBD3 was immunoprecipitated with GFP-nanobody conjugated agarose beads from HEK-293T cells coexpressing SCFD1-HaloTag. Bound SCFD1-HaloTag and the amount of SCFD1-HaloTag in 0.125% of the lysates were detected by direct imaging of the in-gel JF646 HaloTag ligand fluorescence. The amount of GFP and GFP-ACBD3 in the immunoprecipitates was detected with an anti-GFP HRP conjugate antibody. The experiment was repeated three times, and the intensity of the bands was quantified using Fiji. The mean and SD of three independent experiments are shown and a two-tailed t test was performed on the data. ***P ≤ 0.001. (C) SCFD1 is important for the recruitment of PI4KIIIβ to the Golgi apparatus. Widefield imaging of endogenous ACBD3 and PI4KIIIβ in CRISPR-Cas9 KO cells of PI4KIIIβ, ACBD3, and SCFD1. Three biological repeats, nucleus stain = DAPI. Scale bars: 10 μm.
Fig 3: SEC22B and SCFD1 do not interact with MWT374-376 residues in the UR. (A and B) Different truncations of GFP-ACBD3-UR-GOLD domain and GFP-ACBD3 MWT374-376>AAA mutant were co-expressed either with HaloTag-SEC22B (A) or SCFD1-HaloTag (B) in HEK-293T cells and immunoprecipitation experiments were performed as described above. Two biological repeats.
Fig 4: ACBD3 interacts and colocalizes with SCFD1.(A) SCFD1 colocalizes with ACBD3 at the Golgi. Widefield imaging of HeLa cells transfected with SCFD1-HaloTag and stained for endogenous ACBD3. Three biological repeats, nucleus stain = DAPI. Scale bar: 10 μm. (B) ACBD3 interacts with SCFD1. GFP alone or GFP-ACBD3 was immunoprecipitated with GFP-nanobody conjugated agarose beads from HEK-293T cells coexpressing SCFD1-HaloTag. Bound SCFD1-HaloTag and the amount of SCFD1-HaloTag in 0.125% of the lysates were detected by direct imaging of the in-gel JF646 HaloTag ligand fluorescence. The amount of GFP and GFP-ACBD3 in the immunoprecipitates was detected with an anti-GFP HRP conjugate antibody. The experiment was repeated three times, and the intensity of the bands was quantified using Fiji. The mean and SD of three independent experiments are shown and a two-tailed t test was performed on the data. ***P ≤ 0.001. (C) SCFD1 is important for the recruitment of PI4KIIIβ to the Golgi apparatus. Widefield imaging of endogenous ACBD3 and PI4KIIIβ in CRISPR-Cas9 KO cells of PI4KIIIβ, ACBD3, and SCFD1. Three biological repeats, nucleus stain = DAPI. Scale bars: 10 μm.
Fig 5: SEC22B and SCFD1 do not interact with MWT374-376 residues in the UR. (A and B) Different truncations of GFP-ACBD3-UR-GOLD domain and GFP-ACBD3 MWT374-376>AAA mutant were co-expressed either with HaloTag-SEC22B (A) or SCFD1-HaloTag (B) in HEK-293T cells and immunoprecipitation experiments were performed as described above. Two biological repeats.
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