cloning grasp55 gfp  (Addgene inc)

Journal: Journal of Cell Science

Article Title: SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly

doi: 10.1242/jcs.232389

Figure Lengend Snippet: SIRT2 interacts with GRASP55 in mitosis and deacetylates GRASP55 at mitotic exit. (A) SIRT2 co-immunoprecipitates with GRASP55 but not Golgin-84 during mitosis. Asynchronous (AS) and nocodazole-arrested mitotic HeLa cells co-overexpressing GFP-tagged GRASP55 or Golgin-84 and FLAG–SIRT2 were lysed, immunoprecipitated with an anti-GFP antibody, and blotted for GFP and FLAG. The upper bands of FLAG–SIRT2 and GRASP55–GFP in mitotic samples correspond to their phosphorylated forms. (B) Western blots of the HA immunoprecipitation from HEK293T cells expressing HA–SIRT2 and FLAG–GRASP55. (C) In vitro acetylation of GRASP55 with p300 acetyltransferase and deacetylation of GRASP55 by SIRT2. 1.5 µg of GRASP55, 0.15 µg of p300 acetyltransferase and 0.15 or 1.5 µg (as indicated by triangle) of SIRT2 or SIRT2 H150Y were present in the loaded samples. The acetylation level of GRASP55 and p300 was detected by an anti-acetylated lysine (AcK) antibody. (D) Targeted mass spectrometry analysis of GRASP55 K50 acetylation in HEK293T cells transfected with GRASP55–GFP. Mitotic samples were collected after 16 h synchronization with nocodazole. Mitotic exit samples were collected 2 h after release from nocodazole. SIRT2 siRNA was transfected for 72 h. HA–SIRT2 was overexpressed for 24 h. K50 acetylation level was determined as a ratio between modified and unmodified LNK(ac)DNDTLK peptide (n=5). Quantification results are presented as mean±s.e.m. *P<0.05 (two-tailed Student's t-test).

Article Snippet: Mass spectrometry analysis of GRASP55 acetylation GRASP55–GFP was transfected into HEK293T cells and immunoprecipitated using GFP-Trap magnetic beads (Chromotek).

Techniques: Immunoprecipitation, Western Blot, Expressing, In Vitro, Mass Spectrometry, Transfection, Modification, Two Tailed Test

Journal: Journal of Cell Science

Article Title: SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly

doi: 10.1242/jcs.232389

Figure Lengend Snippet: SIRT2 depletion causes Golgi fragmentation and impairs Golgi structure formation. (A–D) Representative immunofluorescence images of HeLa (A) and U2OS (B) cells transfected with control or SIRT2 siRNA and co-stained for GRASP55 (green) and GM130 (red). Representative fragmented Golgi are enlarged in the insets in A. Arrowheads in B indicate cells exhibiting fragmented Golgi. Scale bars: 20 μm. (C,D) Quantification of Golgi fragmentation in A and B from three independent experiments (n=210). (E) SIRT2 overexpression does not significantly perturb the Golgi morphology in U2OS cells. Cells were transfected with FLAG–SIRT2 for 24 h and co-stained for FLAG (green) and GRASP55 (red). Two sets of cells are shown. Scale bar: 20 μm. (F) HeLa cells were transfected with control or SIRT2 siRNA and analyzed by EM. Representative EM images are shown. Scale bar: 500 nm. (G) Quantification of the number of cisternae per stack (n=20). (H) Quantification of the length of Golgi cisternae (n=20). (I) Live-cell images of U2OS cells stably expressing GRASP55–GFP 72 h after SIRT2 siRNA transfection. Note that the Golgi is assembled in control cells (arrows) at 60 min but remains fragmented in SIRT2-depleted cells after 120 min (arrowheads). Scale bar: 20 μm. (J) Quantification of Golgi fragmentation from I in control and SIRT2-knockdown cells using GRASP55–GFP as a marker (n=100 for siControl; n=95 for siSIRT2). All quantification results in this figure are presented as mean±s.e.m. *P<0.05; ***P<0.001 (two-tailed Student's t-test).

Article Snippet: Mass spectrometry analysis of GRASP55 acetylation GRASP55–GFP was transfected into HEK293T cells and immunoprecipitated using GFP-Trap magnetic beads (Chromotek).

Techniques: Immunofluorescence, Transfection, Control, Staining, Over Expression, Stable Transfection, Expressing, Knockdown, Marker, Two Tailed Test

Journal: Journal of Cell Science

Article Title: SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly

doi: 10.1242/jcs.232389

Figure Lengend Snippet: The deacetylase activity of SIRT2 is required for maintaining an intact Golgi structure. (A) HeLa cells were transfected first with SIRT2 siRNA for 48 h, then with a FLAG vector (as control) or siRNA-resistant FLAG–SIRT2 WT or H150Y mutant for another 24 h, and co-stained for GRASP55 (green) and FLAG (red). Scale bar: 20 μm. (B) Cells in A were analyzed by western blotting to show the knockdown efficiency of endogenous SIRT2 and the expression level of FLAG-tagged SIRT2. (C) Quantification of Golgi fragmentation in A from three independent experiments (n=200). Results are presented as mean±s.e.m. **P<0.01 (two-tailed Student's t-test). (D) HeLa cells were transfected with SIRT2 siRNA for 72 h or with FLAG–SIRT2 for 24 h, lysed, and blotted for the indicated Golgi proteins. Control siRNA or an empty FLAG vector were used as controls.

Article Snippet: Mass spectrometry analysis of GRASP55 acetylation GRASP55–GFP was transfected into HEK293T cells and immunoprecipitated using GFP-Trap magnetic beads (Chromotek).

Techniques: Histone Deacetylase Assay, Activity Assay, Transfection, Plasmid Preparation, Control, Mutagenesis, Staining, Western Blot, Knockdown, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly

doi: 10.1242/jcs.232389

Figure Lengend Snippet: GRASP55 deacetylation is required for Golgi structure formation. (A) GRASP55/65 double-knockout cells were transfected with a GFP vector (as control), or GFP-tagged WT, acetylation-deficient mutant K50R or acetylation-mimetic mutant K50Q GRASP55 (labeled G55) for 24 h, and stained for GM130. Note that expression of WT and K50R efficiently rescued the Golgi structure. Scale bar: 20 μm. (B) Cells in A were analyzed by western blotting to show the knockout efficiency of endogenous GRASP55 and the expression level of GFP-tagged GRASP55 WT and mutants. (C) Quantification of Golgi fragmentation in A from three independent experiments (n=210). Quantification results are presented as mean±s.e.m. **P<0.01; ***P<0.001 (two-tailed Student's t-test).

Article Snippet: Mass spectrometry analysis of GRASP55 acetylation GRASP55–GFP was transfected into HEK293T cells and immunoprecipitated using GFP-Trap magnetic beads (Chromotek).

Techniques: Double Knockout, Transfection, Plasmid Preparation, Control, Mutagenesis, Labeling, Staining, Expressing, Western Blot, Knock-Out, Two Tailed Test

Journal: Journal of Cell Science

Article Title: SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly

doi: 10.1242/jcs.232389

Figure Lengend Snippet: GRASP55 deacetylation is required for Golgi reassembly at mitotic exit. (A) GRASP55/65 double-knockout cells were transfected with a GFP vector (as control), or GFP-tagged WT, K50R or K50Q GRASP55 (labeled G55), and stained for GM130. Representative fluorescence images of telophase and cytokinesis cells are shown. Arrowheads indicate cells with fragmented Golgi. Indicated areas, showing the representative Golgi morphology, are enlarged in the insets. Scale bar: 20 μm. (B) Quantification of Golgi fragmentation in A from three independent experiments (n=200). Quantification results are presented as mean±s.e.m. **P<0.01; ***P<0.001 (two-tailed Student's t-test).

Article Snippet: Mass spectrometry analysis of GRASP55 acetylation GRASP55–GFP was transfected into HEK293T cells and immunoprecipitated using GFP-Trap magnetic beads (Chromotek).

Techniques: Double Knockout, Transfection, Plasmid Preparation, Control, Labeling, Staining, Fluorescence, Two Tailed Test

Journal: Journal of Cell Science

Article Title: SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly

doi: 10.1242/jcs.232389

Figure Lengend Snippet: Acetylation-deficient GRASP55 partially rescues Golgi fragmentation caused by SIRT2 depletion. (A) HeLa cells were transfected first with SIRT2 siRNA for 48 h, then with a GFP vector (as control) or GFP-tagged WT, K50R or K50Q GRASP55 (labeled G55) for another 24 h, and stained for GM130. Indicated areas, showing the representative Golgi morphology, are enlarged in the insets. Scale bar: 20 μm. (B) Cells in A were analyzed by western blotting to show the knockdown efficiency of endogenous SIRT2 and the expression level of GFP-tagged GRASP55. (C) Quantification of Golgi fragmentation in A from three independent experiments (n=221). Quantification results are presented as mean±s.e.m. *P<0.05; ***P<0.001 (two-tailed Student's t-test).

Article Snippet: Mass spectrometry analysis of GRASP55 acetylation GRASP55–GFP was transfected into HEK293T cells and immunoprecipitated using GFP-Trap magnetic beads (Chromotek).

Techniques: Transfection, Plasmid Preparation, Control, Labeling, Staining, Western Blot, Knockdown, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly

doi: 10.1242/jcs.232389

Figure Lengend Snippet: GRASP55 deacetylation facilitates self-interaction. HeLa cells were co-transfected with GFP- and FLAG-tagged GRASP55 (G55) WT, K50R or K50Q constructs for 24 h and synchronized with nocodazole for another 18 h. Mitotic cells were collected, lysed, immunoprecipitated with a GFP antibody, and immunoblotted for GFP and FLAG. (A,B) Homologous GFP- and FLAG-tagged GRASP55 constructs were co-transfected, analyzed by co-immunoprecipitation, and their levels quantified. (C,D) Homologous or heterologous GFP- and FLAG-tagged GRASP55 constructs were co-transfected, analyzed, and their levels quantified. (A,C) Western blots of the co-immunoprecipitated proteins. (B,D) Quantification of the interaction efficiency found in experiments shown in A and C as determined by calculating the ratio of FLAG to GFP intensity in the GFP immunoprecipitates from three independent experiments. The homologous interaction of WT GRASP55 or the K50R mutant was normalized to 1 in B and D, respectively. Quantification results are presented as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001 (two-tailed Student's t-test).

Article Snippet: Mass spectrometry analysis of GRASP55 acetylation GRASP55–GFP was transfected into HEK293T cells and immunoprecipitated using GFP-Trap magnetic beads (Chromotek).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Mutagenesis, Two Tailed Test