rabbit anti-wave-2 (d2c8) xp  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: NHSL3 interacts with Ena/VASP proteins and the Scar/WAVE complex and promotes cell migration

doi: 10.1101/2025.04.03.647056

Figure Lengend Snippet: ( A ) Myc-tagged CYFIP2, Nap1, Abi1, Scar/WAVE2, and HSPC300 were co-expressed with NHSL3-EGFP in HEK cells. EGFP-tagged NHSL3 was immunoprecipitated from cell lysates using NHSL3 polyclonal antiserum or normal rabbit IgG as negative control and blots were probed using antibodies against Myc and EGFP. Blot representative of three independent experiments. ( B,C ) Endogenous NHSL3 was immunoprecipitated with NHSL3 polyclonal antiserum or normal rabbit IgG as negative control from B16-F1 lysates, blotted and probed using antibodies against Scar/WAVE2 (B) or Abi1 (C) and NHSL3. Blot representative of 3 independent experiments. ( D-F ) Far western blot experiments using (D) six GST-tagged NHSL3 fragments (Fragment 1 - 6 in (E)) or (F) five sub-fragments of GST-3 (sub-fragment GST-3.1-3.5 in (E)) overlaid with purified MBP-Abi1 or MBP-Abi1ΔSH3 or MBP as negative control. GST-fusion protein containing several Abi SH3 binding motifs of the Lamellipodin protein served as a positive control and GST alone as the negative control. For (D,F) four independent experiments. (E) Schematic overview of the six GST-tagged fragments covering the entire amino acid sequence of NHSL3 and overlapping sub-fragments of fragment 3. The numbering refers to the amino acid numbers at the start and end of each fragment referring to murine NHSL3 isoform c (mmNHSL3 transcript variant 3 with exon1c; Genbank No: NM_001285866).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), Mena mAb clone A351F7D9 (Lebrand et al., 2004); HSC70 (Santa Cruz sc7298).

Techniques: Immunoprecipitation, Negative Control, Far Western Blot, Purification, Binding Assay, Positive Control, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: NHSL3 interacts with Ena/VASP proteins and the Scar/WAVE complex and promotes cell migration

doi: 10.1101/2025.04.03.647056

Figure Lengend Snippet: (A-D) Still images from live cell imaging showing NHSL3-EGFP co-expressed with mScarlet-I-tagged Mena (A) , -Scar/WAVE2 (B) , -Abi1 (C) and - Nap1 (D) in B16-F1 cells. Representative images shown from three independent experiments. Scale bar in (A) for (A-D) represent 20 µm. See also related videos S2-5.

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), Mena mAb clone A351F7D9 (Lebrand et al., 2004); HSC70 (Santa Cruz sc7298).

Techniques: Live Cell Imaging

Journal: bioRxiv

Article Title: NHSL3 interacts with Ena/VASP proteins and the Scar/WAVE complex and promotes cell migration

doi: 10.1101/2025.04.03.647056

Figure Lengend Snippet: ( A ) Myc-tagged Abi1 was co-expressed with wild-type NHSL3-EGFP, EGFP-tagged NHSL3 with the Abi SH3 domain binding site mutated (NHSL3 ΔSH3 -EGFP) or the first and second Ena/VASP binding site mutated (NHSL3 ΔFP41/2 -EGFP). EGFP-tagged NHSL3 was immunoprecipitated from cell lysates using NHSL3 polyclonal antiserum or normal rabbit IgG as negative control and blots were probed using antibodies against Myc and EGFP. Blot representative of three independent experiments. ( B ) Myc-tagged CYFIP2, Nap1, Scar/WAVE2, Abi1, and HSPC300 were co-expressed with wild-type NHSL3-EGFP, EGFP-tagged NHSL3 with the Abi SH3 domain binding site mutated (NHSL3 ΔSH3 -EGFP) or the first and second Ena/VASP binding site mutated (NHSL3 ΔFP41/2 -EGFP) or both NHSL3 ΔSH3ΔFP41/2 -EGFP. EGFP-tagged NHSL3 was immunoprecipitated from cell lysates using NHSL3 polyclonal antiserum or normal rabbit IgG as negative control and blots were probed using antibodies against Myc and EGFP. Blot representative of three independent experiments. (C) HEK cells were co-transfected with Myc-tagged CYFIP1, or CYFIP2, or CYFIP1 with Nap1 or CYFIP2 with Nap1 and with EGFP only as control or EGFP-tagged NHSL3 with the Abi SH3 domain binding site mutated (NHSL3 ΔSH3 -EGFP). EGFP-tagged NHSL3 was pulled down from cell lysates using a nanobody against EGFP and blots were probed using antibodies against Myc and EGFP. Representative blots from three independent experiments (CYFIP1), two independent experiments (CYFIP2), and 6 independent experiments (CYFIP1/NAP1 and CYFIP2/NAP1).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), Mena mAb clone A351F7D9 (Lebrand et al., 2004); HSC70 (Santa Cruz sc7298).

Techniques: Binding Assay, Immunoprecipitation, Negative Control, Transfection, Control

Journal: bioRxiv

Article Title: NHSL3 interacts with Ena/VASP proteins and the Scar/WAVE complex and promotes cell migration

doi: 10.1101/2025.04.03.647056

Figure Lengend Snippet: (A,B) HEK cells were co-transfected with Myc-tagged CYFIP1 or CYFIP2 (A) or Myc-tagged Nap1, CYFIP1 or CYFIP2, Nap1, Scar/WAVE2, Abi1, and HSPC300 (B) and with EGFP only as control or EGFP-tagged NHSL3 with the Abi SH3 domain binding site mutated (NHSL3 ΔSH3 -EGFP) or EGFP-tagged NHSL3 with the Abi SH3 domain binding site and the seven amino acids mediating CYFIP binding mutated (NHSL3 ΔSH3ΔCYFIP -EGFP). EGFP-tagged NHSL3 proteins were pulled down from cell lysates using a nanobody against EGFP and blots were probed using antibodies against Myc and EGFP. Representative blots from three independent experiments. (C,D) Knockout of NHSL3 causes an increase in Arp2/3 complex activity. ( C ) Lifetime images of wild-type (WT) CRISPR Ctrl, NHSL3 CRISPR KO-1, and NHSL3 CRISPR KO-2 B16-F1 cells expressing the Arp2/3 complex biosensor plated on laminin coated glass. Warm colours indicate short lifetimes, and cool colours long lifetimes, of the donor fluorophore mTurq2. Shorter lifetimes represent higher FRET efficiency and therefore higher Arp2/3 complex activity. Representative images from four independent experiments. ( D ) Quantification of average cellular FRET efficiency (E FRET ) which corresponds to Arp2/3 complex activity in WT CRISPR Ctrl (grey circles), NHSL3 CRISPR KO-1 (pink inverted triangles), and NHSL3 CRISPR KO-2 (green diamonds) cells. Data points are the weighted mean FRET efficiency across each individual cell, and bars represent the population mean ± SEM. Data from four independent experiments (N = 4): n = 34 (WT CRISPR Ctrl), n = 37 (NHSL3 CRISPR KO-1), n = 32 (NHSL3 CRISPR KO-2). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test: ∗∗∗∗P < 0.0001 (WT CRISPR Ctrl <> NHSL3 CRISPR KO-1; WT CRISPR Ctrl <> NHSL3 CRISPR KO-2).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), Mena mAb clone A351F7D9 (Lebrand et al., 2004); HSC70 (Santa Cruz sc7298).

Techniques: Transfection, Control, Binding Assay, Knock-Out, Activity Assay, CRISPR, Expressing