Cofilin

Manufactured by Cell Signaling Technology

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Cofilin is a protein that plays a crucial role in the regulation of actin filament dynamics within cells. It is responsible for severing and depolymerizing actin filaments, which is essential for various cellular processes such as cell motility, endocytosis, and cytoskeletal reorganization.

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Anti-cofilin (30 citations) Phospho-cofilin (20 citations) Anti-phospho-cofilin (13 citations) Anti-p-cofilin (8 citations) Anti-phospho-cofilin (Ser3) (7 citations) Phospho-cofilin (S3) (3 citations) Anti-Cofilin (D3F9) (2 citations) Andphospho-COFILIN Ser3 (2 citations) Total- and phospho-cofilin (2 citations) Rabbit monoclonal anti-p-cofilin antibody (2 citations)

77 protocols using cofilin

Analysis of Neuronal Signaling Pathways

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Cell extracts were prepared from neurons treated with or without Tat/TNFα using RIPA buffer (containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1 mM EDTA, 1% NP-40, and protease and phosphatase inhibitor cocktail, Thermo Scientific). Protein concentrations were quantified using the Pierce™ 660 nm protein assay reagent. Equal amounts of protein samples (25 μg/well) were separated on a 4%–12% Bis-Tris polyacrylamide gel, transferred to a nitrocellulose membrane using iBlot 2 NC regular stacks (Thermo Scientific), and probed with antibodies specific for Ca²⁺/calmodulin-dependent protein kinase II (CAMKII) (1:1000, Abcam), phospho-CAMKII (1:1000, Abcam), pP38 (1:1000, Cell Signaling), P38 (1:1000, Cell Signaling), pMEF2A (1:1000, Abcam), MEF2A (1:1000, LifeSpan BioSciences), PINCH (1:1000, BD Biosciences), GAPDH (1:5000, Santa Cruz), ILK1 (1:1000, Abcam), Parvin (1:1000, Cell Signaling), TESK1 (1:1000, Cell Signaling), pCofilin at serine 3 (1:1000, Cell Signaling), Cofilin (1:1000, Cell Signaling), Chronophin (1:1000, Cell Signaling), Tubulin (1:1000, Cell Signaling), mitochondrial Rho GTPase 1 (Miro1) (1:1000, Abcam), KIF5B, kinesin (1:1000, Abcam), or Trak1 (1:1000, ThermoFisher).

Natarajaseenivasan K., Shanmughapriya S., Velusamy P., Sayre M., Garcia A., Gomez N.M, & Langford D. (2020). Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons. Translational Neurodegeneration, 9, 32.

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Protein Analysis of Prostate Cancer Cells

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For protein analysis, RB-proficient and -deficient cells (LNCaP, PC3, LAPC4, C4-2, PC3-ML), RHAMM-overexpressing cells (PC3, LNCaP, PC3-ML), and RHAMM knockdown cells (PC3, PC3-ML, LNCaP) were utilized. Briefly, the cells were harvested by trypsinization, and cell lysis was carried out in RIPA buffer [150 mmol/L NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 50 mmol/L Tris (pH 8.0)] supplemented with protease inhibitors, phosphatase inhibitors, and phenylmethylsulfonyl fluoride. After brief sonication, lysates were clarified, protein concentrations were determined using Bio-Rad Protein Assay Reagent, and an equal amount of protein was subjected to SDS-PAGE and transferred onto Immobilin-P PVDF transfer membranes (Millipore). The membranes were immunoblotted for RB (BD Biosciences), phospho-RB (phospho-serine 780), RHAMM, GAPDH, F-actin (phalloidin, Invitrogen Inc), cofilin, phospho-cofilin (S3) (Cell Signaling Technology), ROCK II, F-actin, lamin B, E-cadherin, vimentin, E2F1, E2F2, and RNRII (Santa Cruz Biotechnology and Abcam) by standard techniques and visualized using Enhanced Western Lightning Chemiluminescence (Perkin-Elmer Life Sciences). The signals were normalized with the internal control lamin B or GAPDH.

Thangavel C., Boopathi E., Liu Y., Haber A., Ertel A., Bhardwaj A., Addya S., Williams N., Ciment S.J., Cotzia P., Dean J.L., Snook A., McNair C., Price M., Hernandez J.R., Zhao S.G., Birbe R., McCarthy J.B., Turley E.A., Pienta K.J., Feng F.Y., Dicker A.P., Knudsen K.E, & Den R.B. (2016). RB Loss Promotes Prostate Cancer Metastasis. Cancer research, 77(4), 982-995.

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Phosphoprotein Analysis of Brain Lysates

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Western blot analysis of brain lysates or of purified phosphoproteins was performed following standard procedures. Briefly, P5 animals were killed and entorhinal cortices from 2 animals (for WT versus PRG-1^−/−) or 3 animals (for WT versus LPA₂-R^−/− or PRG-1^−/−/LPA₂-R^−/−, respectively) were pooled to one biological sample and analyzed as described in the figure legends for the respective experiment. A total of 2.5 mg of protein was then loaded onto the PhosphoProtein purification column (Qiagen). Fractions containing phosphorylated proteins were reduced to 200 µL using Startorius stedim biotech Columns. Protein levels using western blot analysis was performed using following antibodies: CamKI (1:2000; AbCam), Calmodulin (1:1000; Millipore), CamKK (1:500; AbCam), CamKIV (1:1000; Cell Signaling), pCamKI (1:100; provided by Naohito Nozaki and described in Tokumitsu et al., (2004) (link)), LimK1, pLimK1/2, Cofilin and pCofilin (1:1000; Cell Signaling), and beta-actin (1:10.000; MP Biomedicals, LLC). Subsequently, blots were processed for 1 h at room temperature with HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1:5000; Dianova). Densitometric analyzes were performed using ImageJ.

Vogt J., Kirischuk S., Unichenko P., Schlüter L., Pelosi A., Endle H., Yang J.W., Schmarowski N., Cheng J., Thalman C., Strauss U., Prokudin A., Bharati B.S., Aoki J., Chun J., Lutz B., Luhmann H.J, & Nitsch R. (2016). Synaptic Phospholipid Signaling Modulates Axon Outgrowth via Glutamate-dependent Ca2+-mediated Molecular Pathways. Cerebral Cortex (New York, NY), 27(1), 131-145.

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Rac1 Activity Assay Protocol

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Rac1 activity assay was performed according to our previous study 24 (link). Briefly, Rac1-GTP was pulled down with GST-PAK-PBD agarose, and its activity was examined by western blotting using an anti-Rac1 antibody 22 (link), 24 (link), 36 (link). Western blotting were performed according to our previous papers 24 (link), 37 (link), 38 (link). Primary antibodies for western blotting included the following: anti-Rac1 at a dilution of 1:1000 (BD Transduction Laboratories); 1:1000 p-Pak and 1:1000 Pak (Cell Signaling); 1:1000 p-Limk and 1:1000 Limk (Cell Signaling); 1:1000 p-Cofilin and 1:1000 Cofilin (Cell Signaling); Peroxidase-conjugated goat anti-rabbit or anti-mouse IgG second antibodies were purchased from Santa Cruz Biotechnology Inc. and were used at a dilution of 1:5000.

Zhao J., Ying L., Liu Y., Liu N., Tu G., Zhu M., Wu Y., Xiao B., Ye L., Li J., Guo F., Zhang L., Wang H, & Zhang L. (2019). Different roles of Rac1 in the acquisition and extinction of methamphetamine-associated contextual memory in the nucleus accumbens. Theranostics, 9(23), 7051-7071.

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Inhibition of Lck Phosphorylation by Nintedanib

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Cells were seeded onto 96-well plates with 1×10⁵ cells/well, pre-incubated for 20 minutes with nintedanib (10, 30 and 100 nM) and subsequently stimulated with anti-CD3 (10 µg/mL) in combination with anti-CD28 (2 µg/mL) for 5, 10 and 15 minutes. To examine the phosphorylation level of the Lck protein, cells were lysed in cell lysis buffer. Primary antibodies against phospho-Lck-Y394, phospho-Lck-Y505, Lck-total (Life Technologies, Carlsbad, USA) and Cofilin (Cell Signaling Technology, Danvers, USA) were used. The detection was done by a capillary electrophoresis-based protein analysis system (ProteinSimple, San Jose, USA).

Ubieta K., Thomas M.J, & Wollin L. (2021). The Effect of Nintedanib on T-Cell Activation, Subsets and Functions. Drug Design, Development and Therapy, 15, 997-1011.

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Cell Authentication and Culture Conditions

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KHOS and U2OS cells were obtained from American Type Culture Collection (ATCC), and we used short tandem repeat (STR) analysis for the authentication of the two cells at Beijing Microread Genetics Co., Ltd. Cells were cultured in RPMI 1640 medium (HyClone) supplemented with 1% penicillin and streptomycin (Invitrogen) and 10% fetal bovine serum (Gibco) in an environment with a temperature of 37°C and 5% CO2.
The antibodies we used in relevant experiments were as follows: antibodies against p-STAT3, STAT3, p-cofilin, cofilin and VEGFR2, which we purchased from Cell Signaling Technology in United States; anti-PDL2, p-LIMK2, LIMK2 and the RhoA activation assay, which we obtained from Abcam in United States; and anti-GAPDH, which we purchased from Santa Cruz Biotechnology. Y-27632 2HCl was purchased from Selleck.

Zheng B., Zhou C., Qu G., Ren C., Yan P., Guo W, & Yue B. (2020). VEGFR2 Promotes Metastasis and PD-L2 Expression of Human Osteosarcoma Cells by Activating the STAT3 and RhoA-ROCK-LIMK2 Pathways. Frontiers in Oncology, 10, 543562.

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Pancreatic Cancer Cell Cultures

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The pancreatic carcinoma cells Panc-1 and Aspc-1 were purchased from American Type Culture Collection (ATCC (CRL-1469 & CRL-1682)). Normal human pancreatic ductal epithelial cells HPDE6-C7 (HPDE) was kindly provided by Dr. Ming-Sound-Tsao, University of Toronto, Toronto, Canada (18). O-DDHSL and N-dodecanoyl-L-homoserine lactone-3-hydrazone-fluorescein (N-DD-HSL-3-HF) (Fig. 1A & B) were procured from Cayman chemicals, Ann Arbor, MI. N-(3-oxohexanoyl)-L-homoserine lactone (O-HHSL) (Fig. 1C) was purchased from Sigma Chemical Company, St Louis, MO. Antibodies for RhoC, cofilin, IQGAP-1 and β-actin, including an anti-rabbit secondary antibody, were purchased from Cell Signaling Technology (Danvers, MA). All other materials were purchased from Fisher Scientific unless mentioned otherwise.

Kumar A.S., Bryan J.N, & Kumar S.R. (2014). Bacterial Quorum Sensing Molecule N-3-Oxo-Dodecanoyl-L-Homoserine Lactone Causes Direct Cytotoxicity and Reduced Cell Motility in Human Pancreatic Carcinoma Cells. PLoS ONE, 9(9), e106480.

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Evaluation of Cyclovirobuxine D effects

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Cyclovirobuxine D (Cat.no. A0075) was purchased from CHENGDU MUST BIO-TECHNOLOGY CO., LTD (Chengdu, China); Z-VAD-FMK (Cat.no. HY-16658), Mitoquinone (Cat.no. HY-100116A) and CCCP (Cat.no. HY-100941) were purchased from MedChemExpress (Monmouth Junction, NJ, USA); N-acetyl-L-cysteine (NAC) (Cat.no. ST1546) was purchased from Beyotime (Shanghai, China); DMSO (Cat.no. D8418) was purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA); antibodies against C-Caspase3 (Cat.no. 9116S), GAPDH (Cat.no. 2118), phospho -AKT (Cat.no. 4060S) were purchased from Cell Signaling Technology (Beverly, MA, USA); cofilin (Cat.no. ab42824), phospho-cofilin (S3, Cat.no. ab12866), VDAC1 (Cat.no. ab14734), phospho-ERK (Cat.no. ab50011), AKT (Cat.no. ab200195) and ERK (Cat.no. ab17942)were obtained from Abcam (Cambridge, UK); PARP (Cat.no. 13371-1-AP), and cytochrome c (Cat.no. 10993-1-AP) were purchased from Proteintech (Rosemont, IL, USA).

Zhang L., Fu R., Duan D., Li Z., Li B., Ming Y., Li L., Ni R, & Chen J. (2021). Cyclovirobuxine D Induces Apoptosis and Mitochondrial Damage in Glioblastoma Cells Through ROS-Mediated Mitochondrial Translocation of Cofilin. Frontiers in Oncology, 11, 656184.

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Comprehensive Gene and Protein Expression Analysis

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Global mRNA expression profiling by microarray was performed as previously described [15 (link)].
To quantify mRNA and miRNA expression levels by quantitative real-time PCR (qRT-PCR), total RNA was isolated with the Direct-zol RNA MiniPrep Kit (Zymo Research, Irvine, CA, USA). RNA was transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA and miRNA expression was measured in triplicate using TaqMan Gene Expression and MicroRNA Assays (Life Technologies, Carlsbad, CA, USA). For normalization, TBP and RNU6B were used as reference genes for mRNA and miRNA expression analyses, respectively.
For protein analysis, whole cell lysates were prepared with RIPA buffer using equal cell numbers per sample. Protein levels were analyzed using a standard western blot protocol with antibodies against HDGF (AF1606; R&D Systems, Minneapolis, MN, USA), SOX4 (C15310129; Diagenode, Seraing, Belgium), ERK 1/2 (4695; Cell Signaling Technology), phospho-ERK 1/2 (4370; Cell Signaling Technology), cofilin (5175; Cell Signaling Technology), β-actin (3700; Cell Signaling Technology), and α-actinin (4233; Cell Signaling Technology).

Huge N., Reinkens T., Buurman R., Sandbothe M., Bergmann A., Wallaschek H., Vajen B., Stalke A., Decker M., Eilers M., Schäffer V., Dittrich-Breiholz O., Gürlevik E., Kühnel F., Schlegelberger B., Illig T, & Skawran B. (2022). MiR-129-5p exerts Wnt signaling-dependent tumor-suppressive functions in hepatocellular carcinoma by directly targeting hepatoma-derived growth factor HDGF. Cancer Cell International, 22, 192.

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Biochemical Analysis of Sea Cucumber

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Dry sea cucumber C. frondosa was purchased from a local market (Changchun, Jilin Province, China). Antibodies against focal adhesion kinase (FAK) (product code ab131435), phospho-FAK (product code ab81298), paxillin (product code ab32115) and phospho-paxillin (product code ab4833) were purchased from Abcam. Antibodies against p21-activated kinase 1 (PAK1) (cat. no. sc-166887) and phospho-PAK1 (sc-135755) were purchased from Santa Cruz Biotechnology, Inc. LIM domain kinase 1 (LIMK1) (product no. 3842), phospho-LIMK1 (product no. 3841), cofilin (product no. 5175) and phospho-cofilin (product no. 3311) were obtained from Cell Signaling Technology, Inc. ECL chemiluminescent detection reagent and Glutathione Sepharose 4B affinity chromatography resin were obtained from GE Healthcare Life Sciences. Fibronectin, DMSO, EDTA, cysteine and TIRTC-conjugated phalloidin were purchased from Sigma-Aldrich; Merck KGaA. Fetal bovine serum, penicillin, streptomycin and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco Life Technologies; Thermo Fisher Scientific, Inc. All other chemical reagents used in this study were analytical grade.

Zhang M., Chen L., Liu Y., Chen M., Zhang S, & Kong D. (2020). Sea cucumber Cucumaria frondosa fucoidan inhibits osteosarcoma adhesion and migration by regulating cytoskeleton remodeling. Oncology Reports, 44(2), 469-476.

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