Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet: Boc interacts with the WRC (A and D) Boc and tagged WRC constructs were expressed in HEK293T cells as indicated. The lysates were immunoprecipitated with an anti-Boc antibody, and interacting proteins were analyzed through SDS-PAGE and western blot. (B, C, E, F) The relative amount (mean ± SEM) of CYFIP2, NCKAP1, WAVE1, or ABI1 interacting with Boc was calculated by normalizing the amount of corresponding protein in the immunoprecipitate to its amount in the cell lysate and expressed relative to the “Boc + WRC” condition in each experiment. “WRC” refers to the condition in which NCKAP1, CYFIP2, WAVE1, and ABI1 were co-expressed. (B) One-way ANOVA ( p = 0.0001), Tukey’s post hoc test, n = 4 independent experiments. (C, E, F) Unpaired t tests, n = 3 independent experiments for (C) and (F) and n = 4 independent experiments for (E). ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (G) Boc and tagged WRC constructs were expressed in HEK293 cells as indicated. The lysates were immunoprecipitated with an anti-Flag antibody, and interacting proteins were analyzed through SDS-PAGE and western blot. Boc and ABI1-EGFP co-immunoprecipitated with CYFIP2-Flag/NCKAP1-Flag/WAVE1-Flag. See also Figures S1–S3 .

Article Snippet: NAP1-Flag-WT was subcloned from pFLAG-CMV-2-NAP1 to pCAGGS (in Kpn1 at the multiple cloning site (MCS)) using In-Fusion to generate pCAGGS-NCKAP1-Flag. pcDNA3.1(+)Myc-Cyfip2 (mouse) and pCMV-Tag2B-Nap1 (Flag tagged) (mouse) were gifts from Dr. Jia-Jia Liu. pEGFP-C2-Cyfip1 (mouse) was generated by Steffen et al. pcDNA3-Human WAVE1-Flag was a gift from Dr. Greg Bashaw (generated by Westphal et al. ). pCMV6-Entry-BRK1(HSPC300)-Myc-DDK(Flag) was obtained from Origene (cat# RC200804). pCAGGS-NCKAP1 sm -Flag, the shRNA resistant form of human NCKAP1, was made by subcloning NAP1-Flag-WT from pFLAG-CMV-2-NAP1 to pCAGGS (into the Kpn1 site) to have better expression in commissural neurons, along with making silent mutations in NCKAP1, using In-Fusion Cloning Technology.

Techniques: Construct, Immunoprecipitation, SDS Page, Western Blot

Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet: The interaction between Boc and the WRC occurs in live cells and is direct (A–D) The NanoBiT structural complementation reporter system was used to detect interaction between Boc and CYFIP2 in live cells. Boc was fused to the Large BiT (LgBiT) subunit (Boc-LgBiT), and CYFIP2 was fused to the Small BiT (SmBiT) subunit either N-terminally (CYFIP2-SmBiT) or C-terminally (SmBiT-CYFIP2) and expressed in HEK293 or COS7 cells. When the two proteins interact, a luminescent signal is generated in the presence of substrate. (A) Expression of Boc-LgBiT and CYFIP2-SmBit alone or (B) expression of Boc-LgBiT and SmBit-CYFIP2 alone does not generate a luminescent signal. (Left) When Boc-LgBiT is expressed with CYFIP2-SmBiT (A) or SmBit-CYFIP2 (B), they interact to generate a luminescent signal. (Right) Boc-LgBiT also interacts with CYFIP2-SmBiT (A) or SmBit-CYFIP2 (B) in the presence of co-expressed WRC components NCKAP1-Flag, WAVE1-Emerald, ABI1-EGFP, and BRK1-Myc-Flag (“+WRC”). Data are represented as mean ± SEM; error bars representing the SEM are too small to be visible. (Left) Repeated measures one-way ANOVA with Dunnett’s multiple comparison test, (right) paired t test. n = 2–6 independent experiments. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C) Boc lacking an intracellular domain (ICD), BocΔICD-LgBiT, has a significantly lower interaction with CYFIP2-SmBiT and (D) SmBiT-CYFIP2 compared to full-length Boc-LgBiT in the presence of the WRC components NCKAP1-Flag, WAVE1-Emerald, ABI1-EGFP, and BRK1-Myc-Flag. Data are represented as mean ± SEM. Paired t test, n = 3–5 independent experiments. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (E) Schematic of GST-Boc ICD constructs. (F) Coomassie-blue-stained SDS-PAGE gel showing MBP pull-down between purified (MBP) 2 -tagged HSPC300 or WRC and the indicated purified GST-Boc ICD proteins. Only GST-Boc ICD FL and GST-Boc ICD NT are pulled down by (MBP) 2 -WRC.

Article Snippet: NAP1-Flag-WT was subcloned from pFLAG-CMV-2-NAP1 to pCAGGS (in Kpn1 at the multiple cloning site (MCS)) using In-Fusion to generate pCAGGS-NCKAP1-Flag. pcDNA3.1(+)Myc-Cyfip2 (mouse) and pCMV-Tag2B-Nap1 (Flag tagged) (mouse) were gifts from Dr. Jia-Jia Liu. pEGFP-C2-Cyfip1 (mouse) was generated by Steffen et al. pcDNA3-Human WAVE1-Flag was a gift from Dr. Greg Bashaw (generated by Westphal et al. ). pCMV6-Entry-BRK1(HSPC300)-Myc-DDK(Flag) was obtained from Origene (cat# RC200804). pCAGGS-NCKAP1 sm -Flag, the shRNA resistant form of human NCKAP1, was made by subcloning NAP1-Flag-WT from pFLAG-CMV-2-NAP1 to pCAGGS (into the Kpn1 site) to have better expression in commissural neurons, along with making silent mutations in NCKAP1, using In-Fusion Cloning Technology.

Techniques: Generated, Expressing, Comparison, Construct, Staining, SDS Page, Purification

Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet:

Article Snippet: NAP1-Flag-WT was subcloned from pFLAG-CMV-2-NAP1 to pCAGGS (in Kpn1 at the multiple cloning site (MCS)) using In-Fusion to generate pCAGGS-NCKAP1-Flag. pcDNA3.1(+)Myc-Cyfip2 (mouse) and pCMV-Tag2B-Nap1 (Flag tagged) (mouse) were gifts from Dr. Jia-Jia Liu. pEGFP-C2-Cyfip1 (mouse) was generated by Steffen et al. pcDNA3-Human WAVE1-Flag was a gift from Dr. Greg Bashaw (generated by Westphal et al. ). pCMV6-Entry-BRK1(HSPC300)-Myc-DDK(Flag) was obtained from Origene (cat# RC200804). pCAGGS-NCKAP1 sm -Flag, the shRNA resistant form of human NCKAP1, was made by subcloning NAP1-Flag-WT from pFLAG-CMV-2-NAP1 to pCAGGS (into the Kpn1 site) to have better expression in commissural neurons, along with making silent mutations in NCKAP1, using In-Fusion Cloning Technology.

Techniques: Virus, Recombinant, Activity Assay, Protease Inhibitor, Molecular Weight, Concentration Assay, Sequencing, Modification, Affinity Purification, Expressing, shRNA, Generated, Plasmid Preparation, Software, Blocking Assay

Journal:

Article Title: Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold

doi: 10.1093/emboj/19.17.4589

Figure Lengend Snippet: Fig. 10. Oligomerization of WAVE isoforms. HEK-293 cells expressing GFP-tagged WAVE-1 or control vector were co-transfected with FLAG-tagged WAVE-1, WAVE-2 or WAVE-3 (indicated above each lane). Soluble HEK-293 cell extracts were subjected to immunoprecipitation using an anti-FLAG monoclonal antibody. Expression of each WAVE isoform (top panel) was confirmed by immunoblotting with monoclonal antibodies against the FLAG epitope. The migration positions of each WAVE isoform are indicated by arrows. Co-precipitation of GFP-tagged WAVE-1 was detected by immunoblotting using antibodies against GFP (bottom panel).

Article Snippet: Subcloning of human WAVE-1, -2 and -3 into expression vectors encoding N-terminal FLAG tag (FLAG-pCDNA-3; kind gift from Dr Phillip Stork, Vollum Institute, Portland, OR), N-terminal GFP (pEGFP-C1; Clontech), N-terminal histidine tag (pet30a; Novagen) and GST (pGEX-4T1; Pharmacia) was performed following standard restriction digestion and ligation protocols.

Techniques: Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Migration