Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Palmitoylation Controls SynDIG1 Stability, Localization, and Function

doi: 10.1523/JNEUROSCI.4859-14.2016

Figure Lengend Snippet: SynDIG1 palmitoylation is required for clustering and stability in COS cells. A, Hippocampi from 1-month-old mice were lysed and subjected to ABE assay. Palmitoylated (ABE) SynDIG1 and total protein represented by 10% of the input sample in the presence or absence of HAM were measured by immunobloting with anti-SynDIG1 antibodies (SD1). B, Identification of SynDIG1 palmitoylation sites. COS cells were transfected with HA-tagged WT or mutant forms of SynDIG1. After 24 h, cells were lysed and subjected to ABE assay. Palmitoylated (ABE) SynDIG1 and total protein represented by 10% of the input sample in the presence or absence of HAM were measured by immunoblotting with anti-SD1 antibodies. C, Stability of WT and palmitoylation-deficient SynDIG1 mutant C191,192A in COS cells was investigated by treatment with 100 μg/ml CHX for indicated times. Immunoblotting for β-tubulin (β-tub) served as a loading control. D, Graph depicts the percentage of SynDIG1 detected by immunoblotting from lysates isolated after CHX treatment normalized to samples at 0 h. E, SynDIG1 clustering in heterologous cells requires palmitoylation of C192. COS cells were transfected with WT or mutant forms of SynDIG1, fixed after 24 h, and labeled with anti-SD1 (green) and anti-EEA1 (red) antibodies. Bottom, Zoomed-in image of the boxed region in the top panel. Scale bars: E, 10 μm; inset, 2 μm. F, SynDIG1 lacking palmitoylation is retained in the secretory pathway. COS cells were transfected with HA-tagged WT or mutant forms of SynDIG1. After 24 h, cells were treated with BFA or vehicle (Control) for 30 min; fixed; and labeled with anti-HA (green), anti-GM130 (blue), and anti-calreticulin (CR; red) antibodies. Nuclei are indicated by Hoechst stain (magenta) in the merged image. Scale bar, 5 μm.

Article Snippet: The following antibodies were used: mouse anti-SynDIG1 [NeuroMab; immunoblot (IB), 1:1000; immunocytochemistry (ICC), 1:100]; rat anti-HA (Roche; IB, 1:2000; ICC, 1:200); mouse anti-GM130 (BD Transduction Laboratories; ICC, 1:500); rabbit anti-EEA1 (catalog #2900, Abcam; ICC, 1:200); rabbit anti-calreticulin (catalog #2907, Abcam; ICC, 1:600); mouse anti-PSD-95 (NeuroMab; IB, 1:5000; ICC, 1:200); guinea pig anti-VGluT1 (Millipore; ICC, 1:500); mouse anti-β-tubulin (BD Biosciences; IB, 1:10,000); rabbit anti-microtubule-associated protein 2 (MAP2; Sigma-Aldrich; ICC, 1:500); Alexa Fluor 488- and 594-conjugated antibodies (Invitrogen; ICC, 1:200); DyLight 649-, Cy3-, or Cy5-conjugated antibodies (Jackson ImmunoResearch; ICC, 1:200); and HRP-conjugated antibodies (Invitrogen; IB, 1:50,000).

Techniques: Western Blot, Transfection, Mutagenesis, Isolation, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Palmitoylation Controls SynDIG1 Stability, Localization, and Function

doi: 10.1523/JNEUROSCI.4859-14.2016

Figure Lengend Snippet: SynDIG1 localization in neurons requires C192 palmitoylation. A, Hippocampal neurons were transfected at 5 DIV with HA-tagged WT or mutant forms of SynDIG1, fixed at 9 DIV and immunostained with anti-HA (red), anti-EEA1 (green), and anti-GM130 (blue) antibodies. Scale bar, 20 μm. B, C, Graphs represent enrichment of SynDIG1 colocalization with subcellular markers. Similar results were obtained in two independent experiments, n = 10 cells for each condition. Error bars, mean ± SEM. ***p < 0.001. D, Representative stretches of hippocampal neurons treated at 11 DIV with vehicle (DMSO) or 50 μm 2-BP for 4 h; fixed; and stained with antibodies against PSD-95 or SynDIG1 (SD1), VGluT1, and MAP2. Scale bar, 10 μm. E, F, Graphs represent the puncta size and ID of PSD-95 and SD1 upon 2-BP treatment compared with vehicle. Data are the average of two independent experiments, n = 25 cells for each condition. Error bars, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The following antibodies were used: mouse anti-SynDIG1 [NeuroMab; immunoblot (IB), 1:1000; immunocytochemistry (ICC), 1:100]; rat anti-HA (Roche; IB, 1:2000; ICC, 1:200); mouse anti-GM130 (BD Transduction Laboratories; ICC, 1:500); rabbit anti-EEA1 (catalog #2900, Abcam; ICC, 1:200); rabbit anti-calreticulin (catalog #2907, Abcam; ICC, 1:600); mouse anti-PSD-95 (NeuroMab; IB, 1:5000; ICC, 1:200); guinea pig anti-VGluT1 (Millipore; ICC, 1:500); mouse anti-β-tubulin (BD Biosciences; IB, 1:10,000); rabbit anti-microtubule-associated protein 2 (MAP2; Sigma-Aldrich; ICC, 1:500); Alexa Fluor 488- and 594-conjugated antibodies (Invitrogen; ICC, 1:200); DyLight 649-, Cy3-, or Cy5-conjugated antibodies (Jackson ImmunoResearch; ICC, 1:200); and HRP-conjugated antibodies (Invitrogen; IB, 1:50,000).

Techniques: Transfection, Mutagenesis, Staining

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Palmitoylation Controls SynDIG1 Stability, Localization, and Function

doi: 10.1523/JNEUROSCI.4859-14.2016

Figure Lengend Snippet: SynDIG1 palmitoylation is required for clustering and stability in COS cells. A, Hippocampi from 1-month-old mice were lysed and subjected to ABE assay. Palmitoylated (ABE) SynDIG1 and total protein represented by 10% of the input sample in the presence or absence of HAM were measured by immunobloting with anti-SynDIG1 antibodies (SD1). B, Identification of SynDIG1 palmitoylation sites. COS cells were transfected with HA-tagged WT or mutant forms of SynDIG1. After 24 h, cells were lysed and subjected to ABE assay. Palmitoylated (ABE) SynDIG1 and total protein represented by 10% of the input sample in the presence or absence of HAM were measured by immunoblotting with anti-SD1 antibodies. C, Stability of WT and palmitoylation-deficient SynDIG1 mutant C191,192A in COS cells was investigated by treatment with 100 μg/ml CHX for indicated times. Immunoblotting for β-tubulin (β-tub) served as a loading control. D, Graph depicts the percentage of SynDIG1 detected by immunoblotting from lysates isolated after CHX treatment normalized to samples at 0 h. E, SynDIG1 clustering in heterologous cells requires palmitoylation of C192. COS cells were transfected with WT or mutant forms of SynDIG1, fixed after 24 h, and labeled with anti-SD1 (green) and anti-EEA1 (red) antibodies. Bottom, Zoomed-in image of the boxed region in the top panel. Scale bars: E, 10 μm; inset, 2 μm. F, SynDIG1 lacking palmitoylation is retained in the secretory pathway. COS cells were transfected with HA-tagged WT or mutant forms of SynDIG1. After 24 h, cells were treated with BFA or vehicle (Control) for 30 min; fixed; and labeled with anti-HA (green), anti-GM130 (blue), and anti-calreticulin (CR; red) antibodies. Nuclei are indicated by Hoechst stain (magenta) in the merged image. Scale bar, 5 μm.

Article Snippet: The following antibodies were used: mouse anti-SynDIG1 [NeuroMab; immunoblot (IB), 1:1000; immunocytochemistry (ICC), 1:100]; rat anti-HA (Roche; IB, 1:2000; ICC, 1:200); mouse anti-GM130 (BD Transduction Laboratories; ICC, 1:500); rabbit anti-EEA1 (catalog #2900, Abcam; ICC, 1:200); rabbit anti-calreticulin (catalog #2907, Abcam; ICC, 1:600); mouse anti-PSD-95 (NeuroMab; IB, 1:5000; ICC, 1:200); guinea pig anti-VGluT1 (Millipore; ICC, 1:500); mouse anti-β-tubulin (BD Biosciences; IB, 1:10,000); rabbit anti-microtubule-associated protein 2 (MAP2; Sigma-Aldrich; ICC, 1:500); Alexa Fluor 488- and 594-conjugated antibodies (Invitrogen; ICC, 1:200); DyLight 649-, Cy3-, or Cy5-conjugated antibodies (Jackson ImmunoResearch; ICC, 1:200); and HRP-conjugated antibodies (Invitrogen; IB, 1:50,000).

Techniques: Western Blot, Transfection, Mutagenesis, Isolation, Labeling, Staining