Aortic SMC were fixed at 24 hrs after plating by immersion in 2% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS). Cells were then washed in a glycine buffer and incubated overnight at 4 °C with primary antibodies against integrin α2 (Abcam, San Francisco, CA, USA), integrin α5 (Milipore Sigma, Burlington, MA, USA), and smooth muscle α-actin (SMα-actin) (Millipore Sigma, Burlington, MA, USA) diluted in a sodium citrate buffer containing BSA and Triton X [24 (link)]. After washing, cells were incubated with Alexa 568 secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature, washed again and immediately imaged in DPBS. A similar procedure with overnight incubation was followed for the primary antibody against integrin β1 or β3 both pre-conjugated with Alexa 488 (BioLegends, San Diego, CA, USA). For smooth muscle γ-actin (SMγ-actin, Actg2) staining, cells were first fixed with 1% paraformaldehyde in DPBS followed by permeabilization with cold methanol [25 (link)]. Staining was performed as described by using an SMγ-actin primary antibody [26 (link),27 (link)] followed by Alexa 488 secondary antibody (Jackson Immuno Research, West Grove, PA, USA).
Integrin α5
Manufactured by Merck Group
Sourced in United States
Integrin α5 is a cell surface receptor that mediates cell-cell and cell-extracellular matrix interactions. It functions as a receptor for fibronectin, an extracellular matrix protein. Integrin α5 plays a role in various cellular processes, including adhesion, migration, and signaling.
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Lab products found in correlation
Erk1 2, by Cell Signaling Technology (4 mentions)
Integrin β1, by Merck Group (3 mentions)
Integrin α4, by Cell Signaling Technology (2 mentions)
Vinculin, by Cell Signaling Technology (2 mentions)
P pak1, by Cell Signaling Technology (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Anti muc13 mab, by Agilent Technologies (2 mentions)
Anti her2 pab, by Agilent Technologies (2 mentions)
Pher2y1248, by Cell Signaling Technology (2 mentions)
Pfak y925, by Cell Signaling Technology (2 mentions)
Paktth308, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Promega (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Integrin α2, by Abcam (1 mentions)
Alexa 488, by BioLegend (1 mentions)
Alexa 488 secondary antibody, by Jackson ImmunoResearch (1 mentions)
Sm α actin, by Merck Group (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitors, by Dingguo (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
8 protocols using integrin α5
1
Immunofluorescence Staining of Aortic SMCs
2
Western Blot Analysis of Cell Signaling
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Cells were lysed in buffer containing protease and phosphatase inhibitors (Dingguo, Beijing, China). Proteins were quantified, separated by electrophoresis on 8% polyacrylamide gels, and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked and probed with antibodies for integrin α5 (Millipore), integrin β1 (Millipore), ERK1/2 (Cell Signaling, Danvers, MA, USA), phospho-ERK1/2 (Cell Signaling), FAK (04–591; Millipore), Akt (Cell Signaling), p-Akt (Cell Signaling), GSK-3β (Cell Signaling), p-GSK-3β (Cell Signaling), β-catenin (Cell Signaling), and GAPDH (Sigma-Aldrich) overnight at 4 °C, followed by secondary antibodies on the next day. Immunoreactive bands were visualized by chemiluminescence.
3
Cytoskeletal Dynamics in MDA-MB-231 Cells
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MDA-MB-231 cells were seeded in an IBIDI-μSlide (IBIDI, Martinsried, Germany) and cultivated overnight plus treatment period. F-actin was stained with rhodamin-phalloidin (1∶400, R 415, Molecular Pobes/Invitrogen) and nuclei with bisBenzimide H33342 (Sigma-Aldrich, St. Louis, MO, USA). The following antibodies were used: Integrin α5 (Millipore, Upstate), p(S19)-MLC (Cell Signalling Technology, Danvers, MA, USA) and Vinculin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Images were obtained with a Zeiss LSM 510 META (Zeiss, Oberkochen, Germany) or Leica TCS SP8 SMD confocal microscope (Leica, Mannheim, Germany).
4
Wnt Signaling Pathway Regulation
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Proteins extracted from HTR8/SVneo cells were subjected to Western blotting. The membranes were blotted using primary antibodies against human WIF1, N-cadherin (1:1000; Abcam, Cambridge, MA, USA), Wnt5a, β-catenin, integrin β1, integrin α5 (1:1000; Millipore, Darmstadt, Germany), TIMP2, β-actin (1:500; Santa Cruz, Dallas, Texas, USA) at 4°C overnight and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000; Abcam). Densitometric analysis was performed with enhanced chemiluminescence reagents and a Chemi-doc image analyser (Bio-Rad, Hercules, CA, USA).
Co-immunoprecipitation of Wnt5a and WIF1 in HTR8/SVneo cells was examined as previously described [19] . Briefly, cells were lysed in 200 μl of lysis buffer (Beyotime). Lysates were precleared with protein A-agarose (Santa Cruz, Dallas, Texas, USA) and isotype control antibody. The immune complex of 1μg of anti-Wnt5a or WIF1 antibody, protein A-agarose, lysates and binding buffer was then incubated at 4°C overnight. Samples were separated by SDS-PAGE followed by Western blotting using anti-WIF1 or Wnt5a antibody.
Co-immunoprecipitation of Wnt5a and WIF1 in HTR8/SVneo cells was examined as previously described [19] . Briefly, cells were lysed in 200 μl of lysis buffer (Beyotime). Lysates were precleared with protein A-agarose (Santa Cruz, Dallas, Texas, USA) and isotype control antibody. The immune complex of 1μg of anti-Wnt5a or WIF1 antibody, protein A-agarose, lysates and binding buffer was then incubated at 4°C overnight. Samples were separated by SDS-PAGE followed by Western blotting using anti-WIF1 or Wnt5a antibody.
5
Immunoblotting Analysis of Signaling Pathways
6
Immunoblotting Analysis of Signaling Pathways
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The immunoprecipitates were resolved on SDS-PAGE and immunoblotted with anti-MUC13 MAb (produced in our lab # clone PPZ020) and anti-HER2 PAb (catalog number A0485; Dako). The proteins were analyzed by immunoblotting with pHER2Y1248 (catalog number 2247), FAK (catalog number 3285 ), pFAK Y925 (catalog number 3284 ), ERK1/2 (catalog number 9102 ), pERK (catalog number 9101 ), pAKTTh308 (catalog number 2965), AKT (catalog number 9272), PAK (catalog number 2602), pPAK1 (catalog number 2605), Integrin-α4 (catalog number 8440), vinculin (catalog number 13901) α-tubulin (catalog number 2144) that were purchased from Cell Signaling 28 (link). The integrin-α5 (catalog number AB1928) was purchased from EMD Millipore. Mouse (catalog number 4021) and rabbit (catalog number 4011) horseradish peroxidase-conjugated secondary antibodies were purchased from Promega.
7
Focal Adhesion Protein Analysis
8
Integrin Antibody Identification and Characterization
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