U87MG and H4 cells (1 × 104 cells per well) were cultured in 8-well chamber slides (Lab-Tek). Before staining, the cells were starved, activated, or treated with AZD8055 for 24 h. For the knockdown conditions, cells were incubated with siRICTOR, siMAPKAP1, FANA-GSN, and scramble controls for 48 h prior to the steps. Cells were fixed with 4% paraformaldehyde, lysed with 0.2% Triton X buffer, blocked with 1% BSA, and incubated with primary antibodies (anti-RICTOR, anti-MAPKAP1, anti-GSN, anti-β-tubulin (Abcam), anti-mTOR, anti-VIM (Cell Signaling Technologies), anti-FLNA (Millipore), Alexa Fluor 647 Anti-MYH9 (Abcam), Alexa Fluor 488-phalloidin (Invitrogen)) overnight at 4 °C. Samples were stained with secondary antibodies, including Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647 Anti-Rabbit antibodies, and Alexa Fluor 568 Anti-mouse antibody. A Zenon Rabbit IgG labeling kit (Invitrogen) was used to directly conjugate to primary antibodies when necessary. Nuclei of cells were stained with DAPI. Slides were mounted with ProLong anti-fade mountant (Invitrogen). Cells were visualized by an LSM800 with an Airyscan confocal microscope (Zeiss).
Anti flna
Manufactured by Merck Group
Sourced in United States, United Kingdom
Anti-FLNA is a lab equipment product manufactured by Merck Group. It is designed for the detection and quantification of filamin A (FLNA) protein in biological samples. Anti-FLNA utilizes specific antibodies to target and measure FLNA levels, providing researchers with a tool for studying this important cytoskeletal protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti mapkap1, by Abcam (2 mentions)
Anti gsn, by Abcam (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Anti β tubulin, by Abcam (1 mentions)
Anti vim, by Cell Signaling Technology (1 mentions)
Zenon rabbit igg labeling kit, by Thermo Fisher Scientific (1 mentions)
Prolong antifade mountant, by Thermo Fisher Scientific (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Airyscan confocal microscope, by Zeiss (1 mentions)
Anti ect2, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin antibody, by Merck Group (1 mentions)
Anti idh1 r132h antibody, by Dianova (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Anti lpar 1, by Abcam (1 mentions)
Anti lpar 1, by Santa Cruz Biotechnology (1 mentions)
Anti mrtf a, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Motic (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti rac1, by Merck Group (1 mentions)
A11012, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti flna
1
Immunofluorescence Staining of U87MG and H4 Cells
2
Antibody Sources for Cellular Protein Detection
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Rabbit polyclonal anti‐FilGAP antibody was developed as described previously 17 , 19 . Both anti‐FLNa and anti‐integrin β2 antibodies were purchased from Millipore (Billerica, MA). Anti‐ECT2 and anti‐Rac1 antibodies were bought from Santa Cruz Biotechnology (Santa Cruz, CA) and BD Bioscience (San Jose, CA), respectively. Anti‐IDH1 R132H antibody was obtained from Dianova GmbH (Hamburg, Germany). Anti‐α‐tubulin antibody was from Sigma‐Aldrich Chemicals (St. Louis, MO).
3
Duolink in situ Protein-Protein Interaction Analysis
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The DuoLink In situ Red Starter kits mouse/rabbit and goat/mouse (Sigma-Aldrich, Merck, Darmstadt, Germany) were used according to the manufacturer’s protocol for Duolink In situ solutions (Sigma-Aldrich, Merck, Darmstadt, Germany). Anti-MRTF-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FLNA (Millipore, Merck, Darmstadt, Germany), anti-LPAR-1 (Abcam, Cambridge, UK), and anti-LPAR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were used as primary antibodies. Data were visualized with a confocal microscope (Carl Zeiss, Oberkochen, Germany) or a fluorescence microscope (Motic, Wetzlar, Germany), and data were analyzed by 5–15 randomly selected fields.
4
Antibody Protocol for Arl4A/C/D
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The preparation of polyclonal antibodies against Arl4A/C/D was described previously (Li et al., 2007 (link)). The following primary antibodies were used: anti-FLNa (1:1000, catalogue MAB1678; Millipore, Billerica, MA), anti-alpha tubulin (1:5000, catalogue T5168; Sigma, St. Louis, MO), anti-fascin (1:100, catalogue MAB3582; Millipore), anti-FGD6 (1:200, SC-167891; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Rac1 (1:1000, catalogue 05-389; Millipore), anti-Cdc42 (1:250, catalogue 610929; BD Biosciences, San Jose, CA), anti-myc (1:1000, catalogue MMS-150R; Covance, Princeton, NJ), anti-HA (1:1000, catalogue SC-7392; Santa Cruz Biotechnology), anti-LexA (1:1000, catalogue 5397-1; Clontech, Mountain View, CA), and anti–phalloidin-594 and -488 (1:500, catalogue 8953 and 8878; Cell Signaling). 4’,6-Diamidino-2-phenylindole (DAPI) solution was purchased from Millipore (1:5000, catalogue S7113). Horseradish peroxidase–conjugated goat anti-rabbit and anti-mouse antibodies were purchased from GE Healthcare (1:5000, catalogue NA934V and NA931V; Waukesha, WI). Alexa Fluor 594– and 488–conjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Invitrogen (1:500, catalogue A-11012 for Alexa Fluor 594-rabbit; A-11034 for Alexa Fluor 488-rabbit; A-11001 for Alexa Fluor 488-mouse; A-11032 for Alexa Fluor 594-mouse; Grand Island, NY)
5
Collagen Gel Preparation and Immunostaining for Filamin Detection
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Cellmatrix Type I-P (1.6 mg/mL; Nitta Gelatin Inc., Osaka, Japan) was used for preparing collagen gels. Primary antibodies used for the detection of filamins were anti-FLNa (Millipore, Billerica, MA, MAB1680) and anti-FLNb (Abcam, Cambridge, UK, ab97457) antibodies. Primary antibodies for western blotting included anti-di-phosphorylated myosin regulatory light chain (pp-MRLC, Cell Signaling Technology, Beverly, MA, #3674), anti-total amount of myosin regulatory light chain (total-MRLC, Cell Signaling Technology, #3672), anti-total amount of focal adhesion kinase (total-FAK, BD biosciences, San Jose, CA, 610087), anti-phosphorylated focal adhesion kinase at Y397 residue (FAK (pY397), BD biosciences, 611806), and anti-GAPDH antibody (Ambion, Foster City, CA, AM4300). Secondary antibodies of immunofluorescence staining included Alexa-Fluor 546 goat anti-mouse antibody (Molecular probes, Carlscad, CA, A-11003) and Alexa-Fluor 546 goat anti-rabbit antibody (Molecular probes, A-11010). Secondary antibodies for western blot analysis included HRP anti-mouse antibody (Bio-Rad, Hercules, CA, 70-6516) and HRP anti-rabbit antibody (Cell Signaling Technology, #7074). Alexa-Fluor 488 phalloidin (Molecular probes, A-12379) was used for staining F-actin.
6
Western Blot Analysis of Signaling Pathways
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U87MG and H4 cells were seeded into 6-well plates until reaching 80% confluency before the different treatments were performed. For protein extraction, cultured cells were lysed with lysis buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM Tris HCl (pH 7.4), 1 mM EDTA, and EDTA-free protease inhibitor cocktail (PIC) (Roche). Cells were lysed on ice for 15 min and centrifuged at 16,000 × g for 10 min. Total protein concentrations in the whole-cell lysate supernatants were determined by the Bradford protein assay (Bio-Rad). The protein extracts from various samples (25–35 µg) were equally loaded into the SDS-PAGE gel, separated by electrophoresis, and transferred to a nitrocellulose membrane (Bio-Rad). The membranes were blocked in Odyssey® Blocking Buffer (TBS) (LI-COR) for at least 1 h at room temperature or overnight at 4 °C and then probed with primary antibodies overnight at 4 °C. Membranes were washed in TBST and then incubated with IRDye® secondary antibodies (LI-COR). The membranes were scanned on the Odyssey® CLx Imaging Systems (LI-COR). The following antibodies were used: anti-phospho-S6 (S235/236), anti-S6, anti-phospho-AKT (S473), anti-AKT, anti-phospho-MAPKAP1 (T86), anti-mTOR, anti-phospho-FLNA (S2152), anti-MSN, anti-VASP, and anti-CFL (Cell Signaling Technologies), anti-FLNA (EMD Millipore), anti-RICTOR, anti-GSN, anti-MYH9, and anti-MAPKAP1 (Abcam).
7
Protein Immunoprecipitation and Detection
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Anti-FLAG M2 magnetic beads, 3X FLAG peptide, Phalloidin-FITC, anti-Vinculin (VCL), and PP242 were obtained from Sigma-Aldrich. Anti-AU1 agarose beads were from BETHYL Lab. AU1 peptide (DTYRYI) was from COVANCE. Recombinant C-terminal fragment (amino acids 1730–2639) of Human FLNA purified from E. coli was obtained from Creative Biomart. Full-length inactive recombinant AKT1/PKB was from EMD Millipore. Anti-mTOR, Anti-phospho-AKT(Ser473), Anti-AKT, Anti-phospho-FLNA(Ser2152), Anti-phospho-S6(Ser235/236), Anti-S6, Anti-phospho-ERK (Thr202/Tyr204), and Anti-ERK were obtained from Cell Signaling. Anti-FLNA was obtained from EMD Millipore. Anti-RICTOR and anti-SIN1 were obtained from BETHYL Lab. Anti-phospho-CRKII and Anti-CRKII (Ser41) were obtained from Santa Cruz Biotechnology.
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