Clathrin

Manufactured by Cell Signaling Technology

Sourced in United States

Clathrin is a protein that forms a scaffold-like structure on the surface of cells, facilitating the formation of transport vesicles. It plays a crucial role in the process of endocytosis, where materials are brought into the cell from the extracellular environment.

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Lab products found in correlation

13 protocols using clathrin

Biomaterial-based Drug Delivery System

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PHBHHx is kindly provided by BluephaLab, China. Phosphoric acid (H₃PO₄), ethanol (C₂H₅OH), coumarin-6 and hydrochloric acid (HCl) were bought from Shenzhen Tianxiang chemical glass instrument trading company (China). Polyvinyl alcohol(PVA), dimethyl sulphoxide (DMSO), acetone (C₃H₆O), Chloroquine (CQ), bovine serum albumin and 3-methyladenine (3-MA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). N-acryloxysuccinimide (NAS), acrylamide (AAm), N, N, N′, N′-tetramethylethylenediamine (TEMED), N, N-methylene bisacrylamide (BIS), ammonium persulphate (APS), were obtained from Aladdin Industrial Co. LTD. (Shanghai, China). Antibodies against LC3, Arf-6, RhoA, Flotillin, Caveolin, Cdc42, P62, EEA1, Clathrin, were from Cell Signaling Technology. Lyso-Tracker Red and N-(3-Aminopropyl) methacrylamide hydrochloride were from Beyotime Biotechnology (Shanghai, China) and Polymer Science, Inc., respectively.

Sun X., Cheng C., Zhang J., Jin X., Sun S., Mei L, & Huang L. (2019). Intracellular Trafficking Network and Autophagy of PHBHHx Nanoparticles and their Implications for Drug Delivery. Scientific Reports, 9, 9585.

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Exosomal Protein Expression Analysis

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EXOs (pre-, senescent, pre-treated with SB505, and senescent treated with SB505) were collected and immediately lysed and heated for five minutes. Samples were separated using sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher). PVDF membranes were proved using primary antibodies—EEA1, CAV-1, Clathrin, β-actin (Cell Signaling Technology). Bands were visualized using Clarity ECL Western Substrate (BioRad). Full blot images are included in Supplement (Supplementary Figure 4).

Lee A.H., Ghosh D., Koh I.L, & Dawson M.R. (2023). Senescence-associated exosomes transfer miRNA-induced fibrosis to neighboring cells. Aging (Albany NY), 15(5), 1237-1256.

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Subcellular Fractionation and Protein Validation

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To evaluate the subcellular distribution of βARKnt and GRK2 in hearts from βARKnt mice mitochondria-, membrane-, endosomal-enriched and cytosolic fractions were separated using a differential centrifugation method [15 (link)]. βARKnt hearts mice were homogenized in osmotic buffer (25mM Tris HCl; 5 mM EDTA) with protease and phosphatase inhibitors and subjected to a series centrifugation steps including 2,500 rpm to isolate the pellet, 14,000xg for mitochondria, 37,000xg for plasma membrane, 30,000 rpm for the sarcoplasmic reticulum, and 100,000 rpm to isolate endosomes. Pellets were resuspended in lysis buffer (20mM Tris HCl; 1%NP-40, 137mM NaCl 20%glycerol). Protein concentration was measured using a BCA assay (Pierce). These enriched fractions were validated by the predominant presence of corresponding marker proteins, including VDAC1 (mouse monoclonal, Santa Cruz) for mitochondria, Na⁺/K⁺ ATPase (rabbit, Cell Signaling Technology) for membrane, clathrin (rabbit monoclonal, Cell Signaling Technology) for endosomes, and GAPDH (mouse monoclonal, Santa Cruz) for the cytosol.

Bledzka K.M., Manaserh I.H., Grondolsky J., Pfleger J., Roy R., Gao E., Chuprun J.K., Koch W.J, & Schumacher S.M. (2021). A Peptide of the Amino-Terminus of GRK2 Induces Hypertrophy and Yet Elicits Cardioprotection after Pressure Overload. Journal of molecular and cellular cardiology, 154, 137-153.

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Chemotherapeutic Agents and Antibodies

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Doxorubicin and cis-diamminedichloroplatinum (cisplatin; CDDP) were purchased from Selleck Chemicals (Houston, TX). Pacilitaxel was obtained from Calbiochem (Darmstadt, Germany). DMSO was purchased from Sigma. V5-conjugated agarose and the primary antibody against V5 were obtained from Bethyl Laboratories (Montgomery, TX). Anti-P-glycoprotein for flow cytometry was provided by Biolegend (San Diego, CA). Antibodies against GAPDH, Rab5 and Clathrin were obtained from Cell Signaling Technology (Danvers, MA); P-glycoprotein was provided with Merck Millipore (Darmstadt, Germany).

Lee C.Y., Lai T.Y., Tsai M.K., Ou-Yang P., Tsai C.Y., Wu S.W., Hsu L.C, & Chen J.S. (2016). The influence of a caveolin-1 mutant on the function of P-glycoprotein. Scientific Reports, 6, 20486.

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Antibody Staining for Endocytic Markers

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The primary antibodies used in this study were either made in house or were purchased from Cell Signaling Technologies. Stabilin-2 primary antibody was made by the Dr. Paul Weigel lab and was labeled as mAb30,⁶³ (link) and Stabilin-1 primary antibody (clone 911) was a kind gift from Dr. Marko Salmi (University of Turku, Turku, Finland). Anti-mouse immunoglobulin G (IgG) Fab2 Alexa Flour 488 (Cell Signaling Technology, catalog #4408S) was used for detecting these primary antibodies. Primary antibodies for clathrin (catalog #2410S), caveolin 1 (catalog #3238S), early endosomal antigen (EEA1) (catalog #2411S), Rab7 (catalog #9367S), and LAMP1 (catalog #9091S) were purchased from Cell Signaling Technology. Goat anti-rabbit IgG Alexa Fluor Plus 647 (Invitrogen, #A32733) and goat anti-mouse IgG Alexa Fluor 488 (Cell Signaling Technologies, #4408S) secondary antibody was used for detecting these primary antibodies.

Pandey E, & Harris E.N. (2023). Chloroquine and cytosolic galectins affect endosomal escape of antisense oligonucleotides after Stabilin-mediated endocytosis. Molecular Therapy. Nucleic Acids, 33, 430-443.

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Biomimetic Hydrogel for Cellular Uptake

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Lauric acid, bovine serum albumin (BSA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL (Gaithersburg, MD). N-acryloxysuccinimide (NAS), acrylamide (AAm), N,N-methylene bisacrylamide (BIS), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED) and fluorescein isothiocyanate (FITC) were from Aladdin Industrial Co. Ltd. (Shanghai, China). N-(3-Aminopropyl) methacrylamide hydrochloride was purchased from Polymer Science (Monticello, IN). Lipofectamine 2000 was purchased from Life Technologies (Carlsbad, CA). DAPI and Lyso-Tracker Red were from Beyotime Biotechnology (Shanghai, China), Antibodies against LC3, Arf-6, Flotillin, Cdc42, RhoA, P62, EEA1, clathrin, and caveolin were from Cell Signaling Technology, Inc. (Danvers, MA). Antibody against β-actin was obtained from Abmart, Inc. (Shanghai, China). All other materials were commercially available and used as received.

Jiang L., Liang X., Liu G., Zhou Y., Ye X., Chen X., Miao Q., Gao L., Zhang X, & Mei L. (2018). The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery. Drug Delivery, 25(1), 985-994.

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Comprehensive Western Blotting Protocol

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Standard protocols were followed for Western blotting assays [12 (link)]. The primary antibodies used were LAMP1, Tubulin, Dynamin, Clathrin, Caveolae, Na-K ATPase (Cell Signaling Technology, Boston, MA, USA), IL-20R1, IL-20R2, IL-22R1 (Abcam), GRP78 (Santacruz Biotechnology), and MDA-7/IL-24 (Genhunter, Nashville, TN, USA). Secondary antibodies used in this study were from Cell Signaling Technology.

Pradhan A.K., Bhoopathi P., Talukdar S., Das S.K., Emdad L., Sarkar D., Ivanov A.I, & Fisher P.B. (2019). Mechanism of internalization of MDA-7/IL-24 protein and its cognate receptors following ligand-receptor docking. Oncotarget, 10(49), 5103-5117.

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Immunodetection of Epithelial Cytoskeletal Proteins

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Myo1a and Myo1e, 1 μg/ml [Skowron et al. 1998 (link)], Myo1d and sucrase isomaltase, 1.5μg/ml (Santa Cruz Biotechnology, Inc., Dallas, TX), Myo1c and alkaline phosphatase, 1μg/ml (Sigma, St. Louis, MO), villin, 1μg/ml (AMAC, Westbrook, ME), espin, 1μg/ml (Gift from J.R. Bartles, Northwestern University), fimbrin, 1μg/ml [Shibayama et al. 1987 (link)], Myo5a and Myo6, 1μg/ml [Heintzelman et al. 1994 (link)], Myo1a and Myo1e, 1 μg/ml [Skowron et al. 1998 (link)], ezrin, phosphoezrin, and clathrin, 1:1000 (Cell Signaling Technology, Danvers, MA), cytokeratin 8, 1:500 (TROMA-1, DSHB, Iowa City, IA), adaptin beta, 1:500 (BD Biosciences, Franklin Lakes, NJ), NHE3, 1μg/ml (Alpha Diagnostic, San Antonio, TX), NaPi2b 1 μg/ml [Giral et al. 2009 (link)], spectrin, 1:1000 dilution of serum (gift from J. Morrow, Yale School of Medicine). CFTR, AME 4991 CFTR, 1:1000 [Kravtsov et al. 2012 (link)]

Hegan P.S., Kravtsov D.V., Caputo C., Egan M.E., Ameen N.A, & Mooseker M.S. (2015). Restoration of cytoskeletal and membrane tethering defects but not defects in membrane trafficking in the intestinal brush border of mice lacking both myosin Ia and myosin VI. Cytoskeleton (Hoboken, N.J.), 72(9), 455-476.

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Antibody Catalog for Cellular Signaling

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Rabbit antibodies against TLR7, MyD88, NF-κB p65, and phosphorylated IκBα (p-IκBα, Ser32) were purchased from Abcam (Cambridge, United Kingdom). Mouse antibodies against HRS, IRAK1, and c-Myc Tag, goat antibodies to TAB1, and normal rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies against phosphorylated NF-κB p65 (p-p65, Ser536), phosphorylated p38 MAPK (p-p38, Thr180/Tyr182), p38 MAPK, Clathrin, Calnexin, EEA1, Rab5, Rab7, Rab11, LAMP1, Rcas1, and TRAF6 were purchased from Cell Signaling Technology (Beverly, MA). Rabbit antibodies against GFP, PKCζ, β-actin, and GAPDH were purchased from ProteinTech Group (Chicago, IL). Mouse antibody to EV71 VP1 was from Abnova Company (Taipei). Rabbit antibody to EV71 3C was raised against residues 76–88 of 3C protein (Abgent, Suzhou, China). Rat antibodies to FLAG, and anti-mouse CD68 were purchased from BioLegend (San Diego, CA).

Luo Z., Ge M., Chen J., Geng Q., Tian M., Qiao Z., Bai L., Zhang Q., Zhu C., Xiong Y., Wu K., Liu F., Liu Y, & Wu J. (2017). HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection. PLoS Pathogens, 13(8), e1006585.

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Cellular Endocytosis Pathway Analysis

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Bovine serum albumin, dimethyl sulphoxide (DMSO), Chloroquine (CQ), and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-acryloxysuccinimide (NAS), acrylamide (AAm), N, N-methylene bisacrylamide (BIS), ammonium persulphate (APS), N, N, N', N'-tetramethylethylenediamine (TEMED) and Fluorescein isothiocyanate (FITC) were purchased from Aladdin Industrial Co. LTD. (Shanghai, China). N-(3-Aminopropyl) methacrylamide hydrochloride was purchased from Polymer Science, Inc. All other chemicals of the highest quality were commercially available and used as received. Lyso-Tracker Red was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against LC3, Arf-6, Flotillin, Cdc42, RhoA, P62, EEA1, Clathrin, Caveolin were from Cell Signaling Technology. The antibody against LAMP1 was from Santa Cruz Biotechnologies, and the antibody against β-actin was obtained from Abmart.

Zhang J., Zhang X., Liu G., Chang D., Liang X., Zhu X., Tao W, & Mei L. (2016). Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy. Theranostics, 6(12), 2099-2113.

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