wave1  (NeuroMab)

Journal: eLife

Article Title: Endogenous Syngap1 alpha splice forms promote cognitive function and seizure protection

doi: 10.7554/elife.75707

Figure Lengend Snippet: Figure 5. Validation of SynGAP PDZ binding motif (PBM) mutations and construction of the Syngap1PBM mouse line. (A) Schematic diagram for exon map and alternative use of Exon21 in Syngap1 gene. Exon21b encodes for α1 isoform. Exon 21 a encodes for α2 isoform. Point mutations indicated in red alter exon 21b coding sequence without influencing exon21a open reading frame. (B) Schematics of SynGAPα1 and PSD95 domain structure and the location of point mutations. (C) Illustrations of constructs expressed in HeLa cells to study PDZ-dependent interaction between SynGAP and PSD95.

Article Snippet: The lysate was then centrifuged at 35,000 RPM for 30 min at 4 °C and lysate containing 1 mg of protein was incubated with 2 μg Psd95 antibody (Neuromab, catalog # 75–048) at 4 °C overnight with rotation.

Techniques: Biomarker Discovery, Binding Assay, Sequencing, Construct

Journal: eLife

Article Title: Endogenous Syngap1 alpha splice forms promote cognitive function and seizure protection

doi: 10.7554/elife.75707

Figure Lengend Snippet: Figure 7. Characterization of native PSD95 complexes from Syngap1PBM animals. (A) Volcano plot demonstrating the label-free quantitative mass- spectrometry profile of the logarithmic difference in protein levels in the immunoprecipitated PSD95 complexes derived from DIV21 +/+ and PBM/ PBM cultures in inactivated state. Only Gpm6a (shown in black) was significantly altered beyond p > 0.001 cutoff. Blue dots represent proteins with type 1 PDZ-ligands. Green dots represent DLG family proteins. p Values were calculated via t-test for each protein. Samples were derived from individual cultures (4 per genotype) which are immunoprecipitated separately. Log2FC was calculated as ratio of PBM/PBM over +/+. (B) List of proteins that are differentially expressed beyond p > 0.05 cutoff. Note that Iqseq2 and Dlgap3 are PDZ-binding proteins. (C) Mass-spectrometry profile of type-1 PDZ binding motif containing proteins in immunoprecipitated PSD95 complex in +/+ vs PBM/PBM inactivated cultures. (D) Western blots showing relative expression of TARPs and Lrrtm2 in PSD fractions from adult hippocampi in +/+ vs PBM/PBM. (E–G) Quantifications of (D). (E) TARPg8 t(6)=0.6961, P = 0.5124. (F) TARPg2-4 t(6)=0.7924, p = 0.4583 (G) Lrrtm2 t(6)=0.5542, p = 0.5995. Each sample represents hippocampi pooled from 2 mice. (H) Comparison of PSD95 and SynGAP IP complexes as reported by Li et al., 2017 and Wilkinson et al., 2017. Note that PSD95 and SynGAP complexes share diverse range of components involving cytoskeletal and scaffolding proteins.

Article Snippet: The lysate was then centrifuged at 35,000 RPM for 30 min at 4 °C and lysate containing 1 mg of protein was incubated with 2 μg Psd95 antibody (Neuromab, catalog # 75–048) at 4 °C overnight with rotation.

Techniques: Mass Spectrometry, Immunoprecipitation, Derivative Assay, Binding Assay, Western Blot, Expressing, Comparison, Scaffolding

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) Still images from live cell imaging showing NHSL1-EGFP co-expressed with mScarlet-I-tagged Abi1 and (B) Nap1 in B16-F1 cells. Representative images shown from three independent experiments. Scale bar represent 20 µm. Inset is a magnified view of the white box. Scale bar in inset (A) applies to both insets (A,B): 1.25 μm. See also related video S6. (C-E) Endogenous NHSL1 co-localises with Abi1 (C, NHSL1 pAb) Scar/WAVE1 (D, NHSL1 pAb ) , and Scar/WAVE2 (E, NHSL1 mAb ) at the very edge of lamellipodia in B16-F1 mouse melanoma cells. Scale bar in (C) applies also to (D,E): 20 µm. Inset represents a magnified view of the white box. Scale bar in inset in (C) applies also to inset for (D,E): 20 μm. Representative images shown from three independent experiments. See Fig. S8 for line scans of co-localisations in (C) and (E).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Live Cell Imaging

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: Downregulation of CLCa expression alters interaction of clathrin with WAVE during spreading. (A) siRNA-transfected cells were plated on collagen IV for 1 h, fixed and stained with CHC (ab21679) and WAVE2 antibodies. Deconvoluted confocal image planes at the adherent surface are shown with boxed areas magnified on the right to highlight regions of proximity (NC) or separation (KD-a) of clathrin and WAVE2. Object-based image segmentation analysis (Rizk et al., 2014) of whole-cell z-stacks revealed a significant decrease in Pearson coefficients of colocalization in KD-a compared to NC cells [0.564±0.047 and 0.704±0.029 (mean±s.d.), respectively, P<0.0005]. Scale bar: 10 µm. (B,C) Control (B) and CLCa-depleted cells (C) were plated on collagen IV as above and subjected to an in situ proximity ligation assay (PLA) with anti-CHC (green) and -WAVE2 antibodies, as well as DAPI staining of nuclei (blue). Maximum intensity projections of confocal slices in NC cells (three representative fields shown) exhibit PLA-positive loci (red dots). (C) PLA results in KD-a cells (two representative fields). The experiment was performed three times. Scale bars: 10 µm. (D) Number of PLA dots per cell was analyzed by t-test. The box represents the 25–75th percentiles, the whiskers show the range, and the median is indicated. **P<0.005. (E) HeLa cells were lysed 48 h after siRNA transfection and subjected to an immunoprecipitation assay with anti-WAVE1 or control (cIgG) antibodies. The western blot on the right represents 2% of input. The data represent one of three separate experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Expressing, Transfection, Staining, In Situ, Proximity Ligation Assay, Immunoprecipitation, Western Blot