Filamin a

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Filamin A is a highly abundant actin-binding protein that crosslinks actin filaments into orthogonal networks. It functions to maintain the structural integrity of the cytoskeleton and regulate cell shape, migration, and signaling.

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3 protocols using filamin a

Immunofluorescence Staining of Platelet Proteins

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Primary rabbit polyclonal antibodies generated within the HPA project and distributed by Atlas Antibodies (Stockholm, Sweden) were diluted to 2 to 4 µg/ml. A mouse monoclonal anti-alpha tubulin antibody (Ab7291, Abcam, Sweden) diluted to 0.5 µg/ml) and a rat monoclonal anti-KDEL antibody (ab50601, Abcam Sweden) diluted to 2.5 µg/ml were used as common markers for the cytoskeleton and endoplasmic reticulum (ER) respectively. Secondary antibodies goat anti-rabbit IgG Alexa-Fluor 488 (A11034), goat anti-mouse IgG Alexa-Fluor 555 (A21424), and goat anti-rat IgG Alexa-Fluor 647 (A21247) (all from Thermo Fischer Scientific, Waltham, MA) were diluted to 2.5 µg/ml in blocking buffer.
Adherent platelets were stained with antisera against vinculin (Sigma, V9131), filamin A (Santa Cruz Biotechnology, Dallas, TX; sc-17749), non-muscle myosin IIa heavy chain (MYH9, Abcam, Cambridge, UK; ab89837), P-selectin (Santa Cruz Biotechnology; vsc-8419), and reticulon-4 (RTN4, Santa Cruz Biotechnology; sc-271878). Following staining with primary antibodies, platelets were stained with secondary antibodies and TRITC-phalloidin (Sigma, P1951).^{15 (link)}

Bäckström A., Kugel L., Gnann C., Xu H., Aslan J.E., Lundberg E, & Stadler C. (2020). A Sample Preparation Protocol for High Throughput Immunofluorescence of Suspension Cells on an Adherent Surface. Journal of Histochemistry and Cytochemistry, 68(7), 473-489.

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Androgen Receptor Signaling in Cell Adhesion

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Chemicals, unless indicated otherwise, were from Sigma Aldrich (St. Louis, MO, USA). Synthetic androgen R1881 was from Sigma Aldrich, extracellular matrices fibronectin, vitronectin, laminin, collagen I, collagen IV and Src inhibitor PP2 were from Millipore (Billerica, MA, USA). Cycloheximide (Sigma-Aldrich) and MG132 (EMD-Millipore, Billerica, MA, USA) were used at indicated concentrations. Antibodies to AR (N-20), Integrin β1 (M-106) and Filamin A were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Rac-1 (clone 102) was from BD Biosciences (San Jose, CA, USA). Total FAK, phospho-FAK (Y397), total Src, phospho-Src (Y416) were from Cell Signaling, Inc. (Danvers, MA, USA). RNase L monoclonal antibody was kindly provided by Robert Silverman (Cleveland Clinic). Antibodies to β-actin, monoclonal and polyclonal antibodies to Flag tag, Flag-M2 agarose beads were from Sigma Aldrich. Anti-mouse IgG and anti-rabbit IgG HRP linked secondary antibodies were from Cell Signaling, Inc. (Danvers, MA, USA) and ECL reagents were from GE Healthcare (Piscataway, NJ, USA) and Boston Bioproducts (Ashland, MA, USA). Alexa 488-labeled Phalloidin, and Alexa fluor 647 donkey anti-rabbit IgG were from Life Technologies, Carlsbad, CA, USA.

Dayal S., Zhou J., Manivannan P., Siddiqui M.A., Ahmad O.F., Clark M., Awadia S., Garcia-Mata R., Shemshedini L, & Malathi K. (2017). RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells. International Journal of Molecular Sciences, 18(3), 529.

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Comprehensive Protein Analysis via SDS-PAGE

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Lysed samples were loaded onto two 4 to 20% gradient SDS-PAGE gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad) by using standard methods. Gels were blocked with 5% bovine serum albumin in PBS with 0.05% Tween 20 (PBST). Antibodies used for Western blotting included 1:500 Tspan8 (sc-292058, Santa Cruz Biotechnology Inc.), 1:1000 TSG101 (ab83, Abcam), 1:1000 CD9 (ab92726, Abcam), 1:1000 GM130 (sc-55591, Santa Cruz Biotechnology Inc.), 1:1000 HSP70 (sc-32239, Santa Cruz Biotechnology Inc.), 1:1000 galectin-3–binding protein (sc-74970, Santa Cruz Biotechnology Inc.), 1:1000 clusterin (sc-8354, Santa Cruz Biotechnology Inc.), 1:1000 filamin-A (sc-28284, Santa Cruz Biotechnology Inc.), 1:1000 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118L, Cell Signaling Technology), 1:1000 Itsn2 (ab133854, Abcam), and 1:1000 CD49d (PA520595, Invitrogen).

Liu Y., Fan J., Xu T., Ahmadinejad N., Hess K., Lin S.H., Zhang J., Liu X., Liu L., Ning B., Liao Z, & Hu T.Y. (2020). Extracellular vesicle tetraspanin-8 level predicts distant metastasis in non–small cell lung cancer after concurrent chemoradiation. Science Advances, 6(11), eaaz6162.

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