The following antibodies were used in this study: anti-Rac1 (clone 23A8) from Millipore, anti-Cdc42, anti-RhoA and anti-Ezrin from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-Rap1 antibody from Epitomics (Burlingame, CA, USA) and Cell Signaling Technology (Boston, MA, USA), anti-cofilin, anti-phospho-cofilin (Ser3) and anti-phospho-ERM from Cell Signaling Technology, anti- mouse CD11a (clone M17/4) and anti-CD4 from eBioscience (San Diego, CA, USA).
Anti cofilin
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-cofilin is a protein that binds to and regulates the activity of cofilin, an important actin-binding protein involved in the regulation of the actin cytoskeleton. Anti-cofilin can be used to detect and quantify the levels of cofilin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho cofilin, by Cell Signaling Technology (8 mentions)
Anti akt, by Cell Signaling Technology (5 mentions)
Anti phospho cofilin ser3, by Cell Signaling Technology (4 mentions)
Anti erk1 2, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Anti phospho akt, by Cell Signaling Technology (4 mentions)
Anti phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti rhoa, by Cell Signaling Technology (3 mentions)
Anti limk2, by Abcam (3 mentions)
Anti rock1, by Cell Signaling Technology (3 mentions)
Anti cdc42, by Santa Cruz Biotechnology (2 mentions)
Anti rhoa, by Santa Cruz Biotechnology (2 mentions)
Anti ezrin, by Santa Cruz Biotechnology (2 mentions)
Anti phospho erm, by Cell Signaling Technology (2 mentions)
Anti rac1 clone 23a8, by Merck Group (2 mentions)
Anti rap1 antibody, by Abcam (2 mentions)
Anti mouse cd11a clone m17 4, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
30 protocols using anti cofilin
1
Antibody Characterization for Cell Signaling
2
Quantifying RhoA, Rac1, and Cdc42 Activities
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For RhoA activity assay, an immobilized GST-fusion protein, Rhotekin (Millipore, Germany), was used as a probe. The pulled down active RhoA, which binds to the Rho-binding domain of Rhotekin, was detected using anti-RhoA antibody (1:1000). The Rac1 and Cdc42 activities were examined using EZ-Detect™ Rac1 activation Kit (Pierce, USA). Cells were lysed in 25 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 1 mM DTT, and 5% glycerol at 4°C. Active Rac1 was pulled down with GST-fusion protein containing p21-binding domain of p21-activated protein kinase 1 in the presence of immobilized Glutathione Disc. GTP-bound form was detected by western blots with anti-Rac1 or Cdc42 antibodies (1:1000). For p-cofilin assay, equal amounts of lysates from cortical neurons and HEK293 cells were prepared and immunoblotted with anti-cofilin (1:1000, Cell Signaling) and anti-phospho-cofilin (1:1000, Cell Signaling) antibodies.
3
Western Blot Analysis of Cellular Proteins
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Total cellular proteins from individual cells or co-cultured populations were prepared by rinsing cells with cold PBS and scraping with RIPA buffer [20 mM Tris pH 7.4, 150 mM sodium chloride, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 1 mM EDTA, 0.1% (w/v) SDS] in the presence of protease and phosphatase inhibitor cocktails (Roche Diagnostics). 20 μg of protein per well was resolved on SDS-PAGE gels before electro-transfer to PVDF membranes. Following blocking in 5% (w/v) BSA in Tris-buffered saline (TBST), immunoblotting was performed using anti-EphB2 (83029; Cell Signaling Technology), anti-GADPH (ABS16; Millipore), anti-ERK1/2 (9102; Cell Signaling Technology), anti-cofilin (5175; Cell Signaling Technology) or anti-phospho-ERK1/2 (Thr202/Tyr204) (9101; Cell Signaling Technology) antibodies at 1:1000 dilution. Membranes were then washed with TBST and incubated with species-appropriate HRP-conjugated IgG secondary antibodies (Dako, Agilent Technologies) at 1:10,000 dilution. Chemiluminescence was measured using ImageJ after applying Clarity western ECL substrate (BioRad).
4
Quantifying Protein Expression via Western Blot
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Western blot analyses were performed to quantify protein expression in a routine manner [19 (link)20 21 (link)22 (link)]. Primary antibodies were as follow: anti-cJun (1:1,000, monoclonal rabbit; Cell-Signaling Technology, Danvers, MA, USA), anti-phospho-cJun (1:1,000, polyclonal rabbit; Cell-Signaling Technology), anti-Cofilin (1:1,000, monoclonal rabbit; Cell-Signaling Technology), anti-phospho-Cofilin (1:1,000, monoclonal rabbit; Cell-Signaling Technology), and anti-fibronectin (1:2,000, polyclonal rabbit; Abcam, Cambridge, UK). Densitometry was conducted using ImageJ software to analyze the results, reported as relative density of each protein compared to that of β-actin.
5
Western Blot Analysis of BM-OCs
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Day-5 BM-derived OCs (BM-OCs) were lysed in 2× Laemmli sampling buffer, run on SDS–PAGE, and blotted on polyvinylidene fluoride membranes. Primary antibodies were anti-cofilin (1:1000, clone D3F9, 5157; Cell Signaling, Beverly, MA), anti–phospho(Ser-3)-cofilin (1:1000, clone 77G2, 3313; Cell Signaling), anti–β-tubulin (0.3 mg/ml final, T4026; Sigma-Aldrich), anti–Rock-I (1:1000, clone C8F7, 4036; Cell Signaling), anti-Mypt1 (10 μg/ml, A300-888A; Bethyl Laboratories, Montgomery, TX), and anti-p(696)-Mypt1 (1:1000, STA415; Cell Biolabs, San Diego, CA). Enhanced chemiluminescence detection was performed using Amersham ECL Prime Western Blotting Detection Reagent (RPN2232; GE Healthcare, Piscataway, NJ).
6
Quantifying Signaling Pathway Activation
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All primary antibody solutions were prepared in TBST containing 5% bovine serum albumin at a 1:1000 dilution, except otherwise stated. The following primary antibodies were used: anti-phospho-mTOR (Ser2448) and anti-mTOR (both from Cell Signaling), anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-ERK1/2 (both at 1:2000; Cell Signaling), anti-phospho-Akt (Ser473 and Thr308) and anti-Akt (all from Cell Signaling), anti-phospho-p70S6K (Thr389 from Millipore and Thr421/Ser424 from Cell Signaling), anti-p70S6K (Cell Signaling), anti-phospho-cofilin (Ser3, Abcam) and anti-cofilin (Cell Signaling), anti-phospho-PAK1/2/3 (Invitrogen) and anti-PAK3 (1:500, Millipore), anti-spectrin (1:2000; Millipore), anti-Arc (1:5000; Millipore), anti-actin (1:10000; Millipore), and anti-PTEN (Cell Signaling).
7
HeLa Cell Protein Immunodetection
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HeLa cells were obtained from ATCC and maintained in DMEM with 10% FBS. Anti-GST antibody was from Santa Cruz (Santa Cruz, CA), anti-HA, anti-FLAG (M2) and anti-β-actin from Sigma-Aldrich (St Louis, MO), anti-GFP/YFP (Ab290) from Abcam (Cambridge, MA), anti-PKD3 and anti-SSH1L from Bethyl Laboratories (Montgomery, TX), anti-pS744/748-PKD (recognizes pS706/710 for human PKD2 and pS731/735 for human PKD3), anti-LIMK, anti-pT508-LIMK, anti-PAK4, anti-pS474-PAK4, anti-Cofilin and anti-pS3-Cofilin from Cell Signaling Technology (Danvers, MA), anti-PKD2 from Millipore (Billerica, MA). The monoclonal antibodies directed against PKD1 and pS978-SSH1L were described and characterized previously [20] (link). Secondary HRP-linked antibodies were from Millipore. Secondary antibodies for immunofluorescence analysis were goat-anti-mouse Alexa Fluor 488 F(ab’)2 and goat-anti-rabbit Alexa Fluor 488 F(ab’)2 from Invitrogen (Carlsbad, CA). TransIT HeLa-Monster (Mirus, Madison, WI) was used for transient transfection of cells.
8
Imatinib Protocol for Cell Signaling
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Imatinib was purchased from Selleckchem company (Houston, USA). After being dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA), it was subpackaged in different concentrations and stored at -20℃. The following antibodies were used: anti-cofilin, anti-p-cofilin (Ser-3), and anti-GAPDH, which were all from Cell Signaling Technology (Beverly, MA, USA). Anti-Arp2, anti-Arp3 were from Abcam (Cambridge, UK).
9
Eupatilin Inhibits RANKL-Induced Osteoclastogenesis
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Male, 5-week-old ICR mice were purchased from Damul Science (Daejeon, Korea). Ten-week-old OVX mice were purchased from Central Lab. Animal Inc. (Seoul, Korea). Mice were kept in a temperature (22 °C–24 °C) and humidity (55%–60%) controlled environment with a 12 h light/dark cycle. All experiments were performed in accordance with guidelines for animal experimentation of the Institute Committee of Wonkwang University. Eupatilin was obtained from DONG-A PHARM (Seoul, Korea). Soluble, recombinant human M-CSF and human RANKL were obtained from PeproTech EC, Ltd. (London, UK). Anti-Akt, anti-phospho-Akt, anti-GSK3β, anti-phospho-GSK3β, anti-IκB, anti-phospho-IκB, anti-Cofilin, anti-phospho-Cofilin, anti-p38, anti-phospho-p38, anti-ERK, anti-phospho-ERK, anti-JNK, and anti-phospho-JNK antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-c-Fos, and anti-NFATc1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A monoclonal β-actin antibody was obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), α-minimum essential medium (α-MEM), and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). All other chemicals were of analytical grade or complied with the standards required for cell culture.
10
Antibody Validation for Immunoblotting
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The following antibodies were used in this study (with dilution factor for immunoblotting): anti-PKN2 (#2612, 1:1,000), anti-YAP (#14074, 1:1,000), anti-YAP/TAZ (#8418, 1:1,000), anti-phospho-YAP (Ser127) (#4911, 1:1,000), anti-phospho-EGFR (Tyr1068) (#3777, 1:1,000), anti-EGFR (#4267, 1:20,000), anti-phospho-AKT (Ser473) (#4058, 1:1,000), anti-AKT (#9272, 1:5,000), anti-phospho-ERK1/2 (Thr202/Tyr204) (#9101, 1:1,000), anti-ERK1/2 (#9102, 1:5,000), anti-ARIH2/TRIAD1 (#13689, 1:1,000), anti-BTAF1 (#2637, 1:1,000), anti-GNAQ (#14373, 1:1,000), anti-HSP90 (#4877, 1:5,000), anti-ALDOA (#8060, 1:5,000), anti-METAP2 (#12547, 1:1,000), anti-GAPDH (#2118, 1:5,000), anti-RHOA (#2117, 1:1,000), anti-Cofilin (#5175, 1:1,000), anti-phospho-Cofilin (Ser3) (#3313, 1:1,000), from Cell Signaling Technology; anti-α-Tubulin (T6074, 1:20,000) and anti-β-Actin (A1978, 1:20,000) from Sigma; anti-GNB2 (ab81272, 1:1,000), anti-RIC8A (ab97808, 1:1,000), anti-USP22 (ab195289, 1:1,000), anti-CUL5 (ab184177, 1:1,000), anti-RNF7 (ab181986, 1:1,000), anti-PDCD10 (ab180706, 1:1,000), from Abcam; anti-KCTD5 (#15553–1-AP, 1:1,000), anti-PSAT1 (#10501–1-AP, 1:5,000), from Proteintech; Goat Anti-Rabbit IgG Antibody, (H+L) HRP conjugate (#AP307P, 1:5,000), Goat Anti-Mouse IgG Antibody, (H+L) HRP conjugate (#AP308P, 1:5,000), from Millipore Sigma.
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