plasmids  (Addgene inc)

Journal: Cell communication and signaling : CCS

Article Title: Hax1 regulate focal adhesion dynamics through IQGAP1.

doi: 10.1186/s12964-023-01189-y

Figure Lengend Snippet: Fig. 1 Identification of IQGAP1 as a novel Hax1 Binding Partners by Tandem Affinity Purification. A, Hax1 and its associated proteins were isolated by tandem affinity purification and resolved by SDS-PAGE. Immunoglobulin G heavy chain and light chain are marked by arrowheads. The putative band for Hax1 is marked by star. Putative band for IQGAP1 is marked by an arrow. B, IQGAP1 were found as Hax1 binding protein by mass spectrometric sequencing of proteins from the coomassie blue-stained gel. Identified matched peptides were shown in bold red. C, HEK293T cells were transfected with different plasmids as indicated. Cell lysates were immunoprecipitated (IP) with HA-agarose beads and immunoblotted (IB) with different antibodies as indicated. For whole-cell lysate (WCL), 10 μg of total protein was used. Note that only Hax1 specifically pulls down IQGAP1. D, Hax1 immunoprecipitates from MCF7 cells were immunoblotted with antibodies against IQGAP and Hax1. For whole-cell lysate (WCL), 10 μg of total protein was used. Note that Hax1 immunoprecipitation brought down IQGAP1. E, MCF7 cells were immunostained for IQGAP1 (red), Hax1 (green), and DAPI (blue). Scale bar, 10 μm. Cells were imaged by Nikon’s Structured Illumination microscope. The boxed areas are magnified as insets. Note that Hax1 colocalizes with IQGAP1 signal in the protrusive region and accumulates at the leading edge of migrating cells

Article Snippet: The plasmid encoding full length IQGAP1 Cdna: pcDNA3-Myc-IQGAP1 was a gift from David Sacks (Addgene plasmid #30,118; http:// n2t. net/ addge ne: 30118; RRID:Addgene_30118) [30].

Techniques: Binding Assay, Affinity Purification, Isolation, SDS Page, Sequencing, Staining, Transfection, Immunoprecipitation, Microscopy

Journal: Cell communication and signaling : CCS

Article Title: Hax1 regulate focal adhesion dynamics through IQGAP1.

doi: 10.1186/s12964-023-01189-y

Figure Lengend Snippet: Fig. 3 Hax1 recruits IQGAP1 to microtubules and the leading edge of migrating cells. A, MCF7 cells were transfected with DsRed-Hax1 and labeled with antibodies against tubulin (green), and IQGAP1 (magenta). Scale bar, 10 μm. Note that white arrows indicate the membrane ruffle-like localization of Hax1 is superimposed onto that of the IQGAP1 signal and the three channels are merged. B, Immunofluorescence staining for tubulin (green) and IQGAP1 (red) in MCF7 cells transfected with the indicated siRNAs and followed with or without treatment of nocodazole (10 μM) for 30 min. Cells were imaged by super-resolution N-SIM microscopy. The boxed areas are magnified as insets. Scale bar, 10 μm. C, Co-localization of IQGAP1 and MT (tubulin) was determined by Pearson correlation coefficient (n = 20 cells for each). Note that Hax1 localization with MT is reduced significantly in both nocodazole treated cells and Hax1-depleted cells (p < 0.0001). D, The interaction with microtubules (MT) was examined by co-sedimentation assay. The presence of proteins in the original lysate (Input), pellet of ultracentrifugation, as well as the supernatant (Sup) was determined by immunoblot with anti-IQGAP1 antibody

Article Snippet: The plasmid encoding full length IQGAP1 Cdna: pcDNA3-Myc-IQGAP1 was a gift from David Sacks (Addgene plasmid #30,118; http:// n2t. net/ addge ne: 30118; RRID:Addgene_30118) [30].

Techniques: Transfection, Labeling, Membrane, Immunofluorescence, Staining, Microscopy, Sedimentation, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: Hax1 regulate focal adhesion dynamics through IQGAP1.

doi: 10.1186/s12964-023-01189-y

Figure Lengend Snippet: Fig. 4 Suppression of Hax1 alters the subcellular location of IQGAP1 at FAs. A, Representative immunofluorescence images of mock or Hax1 knockdown MCF7 cells stained for Vinculin (green) and IQGAP1 (red). Cells were imaged by super-resolution N-SIM microscopy. The boxed areas are magnified as insets. White arrows indicate accumulation of IQGAP1 at the leading edge in control cells. Red arrows point at IQGAP1 localization at vinculin-positive FAs in control cells. Scale bar, 10 μm. B, Co-localization of IQGAP1 and FA (vinculin) was determined by Pearson correlation coefficient (n = 20 cells for each). Note that IQGAP1 co-localization with vinculin at the rim of the leading edge is reduced significantly in Hax1-depleted cells (p < 0.0001). C, Presence of IQGAP1 and Hax1 in isolated FAs or WCL (20 μg) from mock or Hax1 KD cells was determined by immunoblots. D, Protein abundance in isolated FAs or WCL (20 μg) from cells treated with or without nocodazole was determined by immunoblot with different antibodies as indicated

Article Snippet: The plasmid encoding full length IQGAP1 Cdna: pcDNA3-Myc-IQGAP1 was a gift from David Sacks (Addgene plasmid #30,118; http:// n2t. net/ addge ne: 30118; RRID:Addgene_30118) [30].

Techniques: Immunofluorescence, Knockdown, Staining, Microscopy, Control, Isolation, Western Blot, Quantitative Proteomics

Journal: Cell communication and signaling : CCS

Article Title: Hax1 regulate focal adhesion dynamics through IQGAP1.

doi: 10.1186/s12964-023-01189-y

Figure Lengend Snippet: Fig. 5 The IQGAP1-Hax1 interaction regulates cell migration. A, In MCF7 cells, suppression of Hax1, IQGAP1, or Hax1/IQGAP1 exhibits the same defect in directional migration. The migration of confluent monolayers of mock, Hax1 -depleted, IQGAP1-depleted, or Hax1/IQGAP1-depleted MCF7 cells were scratched, followed by visualization with phase-contrast microscopy at the indicated time points. B, Characterization of the efficiency and specificity of various siRNA oligonuclotides in MCF7 cells. The protein levels of Hax1, IQGAP1, or Hax1/IQGAP1 were significantly decreased in MCF7 cells transfected with corresponding siRNAs. C, The kinetics of in vitro wound healing in A are quantified. Note that suppression of Hax1, IQGAP1 or Hax1/IQGAP1 in MCF7 cells all lead to significant delay of in vitro wound healing (n = 3, p < 0.0001, Student’s t test). D, In MCF7 cells, directional migration of Hax1 knockdown cells and Hax1 –depleted cells rescued with different truncation mutants of Hax1 were evaluated by in vitro scratch wound assays. The migration of confluent monolayers of mock, Hax1 -depleted, or MCF7 cells co-transfected with Hax1 siRNA and two HA-Hax1 constructs (HA-Hax1-NT and HA-Hax1-CT) were scratched, followed by visualization with phase-contrast microscopy at the indicated time points. E, The kinetics of in vitro wound healing in D are quantified. Note that re-expression of Hax1-CT but not Hax1-NT mutant in Hax1-depleted MCF7 cells successfully rescues the defect of in vitro wound healing (n = 3, p < 0.01, Student’s t test)

Article Snippet: The plasmid encoding full length IQGAP1 Cdna: pcDNA3-Myc-IQGAP1 was a gift from David Sacks (Addgene plasmid #30,118; http:// n2t. net/ addge ne: 30118; RRID:Addgene_30118) [30].

Techniques: Migration, Microscopy, Transfection, In Vitro, Knockdown, Construct, Expressing, Mutagenesis