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Gm130

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GM130 is a membrane-bound protein that functions as a cis-Golgi matrix protein. It plays a role in the organization and architecture of the Golgi apparatus. GM130 is involved in the docking and tethering of transport vesicles to the Golgi complex.

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Lab products found in correlation

Tsg101, by Santa Cruz Biotechnology (3 mentions) Calnexin, by Santa Cruz Biotechnology (3 mentions) Bca kit, by Thermo Fisher Scientific (3 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Dynein heavy chain, by Santa Cruz Biotechnology (1 mentions) Mouse monoclonal alpha tubulin, by Developmental Studies Hybridoma Bank (1 mentions) Mouse monoclonal nagk, by Santa Cruz Biotechnology (1 mentions) Ankyrin g, by Santa Cruz Biotechnology (1 mentions) Hsp70, by Santa Cruz Biotechnology (1 mentions) Image lab v6, by Bio-Rad (1 mentions) Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Alpha tubulin, by Developmental Studies Hybridoma Bank (1 mentions) Peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions) Bradford assay, by Merck Group (1 mentions) Las 4000, by Fujifilm (1 mentions) 4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions) Mitofilin, by Abcam (1 mentions) Dsdna, by Santa Cruz Biotechnology (1 mentions)

12 protocols using gm130

1

Antibody Dilutions for Protein Localization

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The following antibodies were used at the indicated dilutions unless otherwise mentioned: chicken polyclonal NAGK (1:1000 for ICC; GenWay Biotech, Inc., now GW22347, Sigma, USA); mouse monoclonal NAGK (1:10 for PLA; Santa Cruz Biotechnology Inc., Dallas, TX); rabbit polyclonal NAGK (1:50 for PLA; GeneTex, USA); rabbit polyclonal DYNLRB1 (1:25; Proteintech Group, Inc., USA), rabbit polyclonal dynein heavy chain (1:25; Santa Cruz); mouse monoclonal TGN38 (1:50; BD Biosciences, USA); rabbit polyclonal GM130 (1:25; Santa Cruz); mouse monoclonal alpha tubulin (1:10; broth preparation, Developmental Studies Hybridoma Bank, University of Iowa, USA); and mouse monoclonal Ankyrin G (1:50, Santa Cruz). The plasmids used for transfection were pDsRed2 vector, pDsRed2-tagged wild-type NAGK, pEGFP-tagged point mutants of NAGK (-N36A, -D107A, and -C143S), and NAGK short hairpin (sh) RNA, all as previously described (Lee et al., 2014a (link); 2014b (link)).
Islam M.A., Sharif S.R., Lee H, & Moon I.S. (2015). N-Acetyl-D-Glucosamine Kinase Promotes the Axonal Growth of Developing Neurons. Molecules and Cells, 38(10), 876-885.
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2

EV Protein Quantification and Western Blot Analysis

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Protein concentration was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer’s instructions. Ten micrograms of EVs and cells were lysed with 2× Laemlli buffer with β-mercaptoethanol and denatured by heating at 70 °C for 5 min. Samples were electrophoresed on a 4%–12% TGX stain-free gel (Bio-Rad, Irvine, CA, USA) at 200 Volts for 30–40 min. Samples were then blotted onto a nitrocellulose membrane using the wet transfer method overnight at 100 volts. After blotting, membranes were blocked for 2 h in 5% semi-skimmed milk in Tris-buffered saline with Tween (TTBS) followed by incubation in primary antibodies Alix (1:200), Hsp70 (1:200), GM130 (1:200) (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at RT. After three five-minute washes, the membrane was probed with a goat anti-mouse IgG-HRP conjugated secondary antibody (1:1000 in TTBS, Life Technologies Limited, Paisley, UK). The HRP-conjugated secondary antibody was added for 1 h, and the wash step was repeated. SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was added to the membrane and imaged with ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA) using Image Lab v.6.0.1 software (Bio-Rad, Hercules, CA, USA).
Hyland M., Mennan C., Wilson E., Clayton A, & Kehoe O. (2020). Pro-Inflammatory Priming of Umbilical Cord Mesenchymal Stromal Cells Alters the Protein Cargo of Their Extracellular Vesicles. Cells, 9(3), 726.
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3

Immunocytochemistry and Proximity Ligation Assay

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The following antibodies were used at the indicated dilutions unless otherwise indicated: chicken polyclonal NAGK (1:1000 for ICC, GenWay Biotech Inc., San Diego, CA, USA, now GW22347, Sigma, St. Louis, MO, USA); mouse monoclonal NAGK (1:10 for PLA; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal NAGK (1:50 for PLA; GeneTex, Irvine, CA, USA); rabbit polyclonal DYNLRB1/LC7 (1:50 for ICC and 1:25 for PLA; Proteintech Group, Chicago, IL, USA); rabbit polyclonal dynein light-chain 1/LC8 (1:50 for PLA; Santa Cruz); rabbit polyclonal dynein heavy chain (DHC; 1:50 for ICC and 1:25 for PLA; Santa Cruz); rabbit affinity-isolated kinesin 5B (1:200 for ICC; Sigma); mouse monoclonal DDK (1:100 for PLA; Origene Technologies, Rockville, MD, USA); mouse monoclonal TGN38 (1:50; BD Biosciences, San Jose, CA, USA); rabbit polyclonal GM130 (1:25; Santa Cruz); and mouse monoclonal alpha-tubulin (1:10; broth preparation, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). The plasmid used for transfection was pCMV6-Myc-DDK-tagged rat NAGK (Origene).
Islam M.A., Sharif S.R., Lee H., Seog D.H, & Moon I.S. (2015). N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points. Experimental & Molecular Medicine, 47(8), e177-.
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4

Dot Blot Analysis of Extracellular Vesicles

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MSCs and MSC-derived EV extracts were lysed using RIPA lysis and extraction buffer with added proteinase inhibitor and phosphatase inhibitor cocktails (Sigma). Protein concentration was measured using a Bradford assay (Sigma). For dot blot analysis [37 (link)], 10 μg of protein from each sample were added onto nitrocellulose membranes and dried for 10 min. The membranes were blocked using 1% skimmed milk in TBS-T (0.1% Tween20 (Sigma) in Tris buffer) and incubated with primary antibodies against CD63, TSG101, or GM130 (Santa Cruz) for 30 min. Membranes were then washed with TBS-T, incubated with peroxidase conjugated secondary antibody (vector laboratories) for 30 min, and then washed with TBS-T, before a 1-minute incubation with ECL detection reagent. Membranes were immediately imaged using LAS-4000 (Fujifilm).
Nizamudeen Z., Markus R., Lodge R., Parmenter C., Platt M., Chakrabarti L, & Sottile V. (2018). Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging. Biochimica et Biophysica Acta. Molecular Cell Research, 1865(12), 1891-1900.
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5

Immunofluorescence Analysis of H9C2 Cells

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The experiment was carried out as described (Haag et al. 2018 (link)). Briefly, H9C2 were grown to a certain density on a 6-well plate overlay. Then, the cells were fixed in 4% paraformaldehyde and drilled with 0.2% Triton and blocked with 5% bovine serum albumin (BSA, Solarbio, China). Subsequently, the cells were incubated with the primary antibodies including mitofilin (1:100, Abcam, UK), dsDNA (1:100, Santa Cruz, USA), STING (1:100, CST, USA), GM130 (1:100, Santa Cruz, USA), and p65 (1:100, CST, USA) overnight at 4 C, and then incubated with FITC- or CY3-bound secondary antibodies for 1 h. 4,6-Diamidino-2-phenylindole (DAPI, Abcam, UK) was used for nuclear staining. Finally, cells were observed by a confocal microscope (Leica, Germany).
Ma X.M., Geng K., Law B.Y., Wang P., Pu Y.L., Chen Q., Xu H.W., Tan X.Z., Jiang Z.Z, & Xu Y. (2022). Lipotoxicity-induced mtDNA release promotes diabetic cardiomyopathy by activating the cGAS-STING pathway in obesity-related diabetes. Cell Biology and Toxicology, 39(1), 277-299.
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6

Immunofluorescence Assay for Cellular Markers

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Immunofluorescence assays were carried out as described previously [18 (link)]. Cells were fixed in 4% paraformaldehyde at room temperature for 20 min, permeabilized with 0.3% Triton X-100 for 10 min, and subsequently blocked with PBS containing 5% goat serum. Cells were incubated overnight with primary antibody P65 (Cell Signaling Technology, Boston, USA), GP73 (Abcam, Cambridge, UK), or GM130 (Santa Cruz Biotechnology, CA, USA) at 4 °C, followed by secondary antibodies tetramethylrhodamine (TRITC)-conjugated goat anti-mouse immunoglobulin G (IgG) (Origene, MD, USA) or fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Origene, MD, USA) for 1 h at room temperature. Lastly, the samples were stained with Hoechst (10 μg/mL) at room temperature for 10 min and photographed with a confocal laser microscope (Leica TCS-SP8, Heidelberg, Germany).
Yang D., Yao M., Yan Y., Liu Y., Wen X., Chen X, & Lu F. (2021). Deoxycholic Acid Upregulates Serum Golgi Protein 73 through Activating NF-κB Pathway and Destroying Golgi Structure in Liver Disease. Biomolecules, 11(2), 205.
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7

Evaluating Extracellular Vesicle Purity

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A in-well western was performed to confirm EV purity. Briefly, 1×106 EVs were fixed in 2% PFA for 10 minutes, followed by washing with PBS once. EVs were then blocked in PBS solution containing 3% BSA for 1 h at room temperature. After washing twice with PBS solution, EVs were incubated with Alex Fluor 647-labeled CD81(1:200, ThermoFisher) and GM130 (1: 200, Santa Cruz) overnight at 4°C, followed by secondary antibody incubation for 1 h at room temperature. EVs were washed twice with PBS, resuspended in 150 μL of PBS, and loaded to a 96-well plate. The fluorescent imaging was performed using LI-COR ODYSSEY CLx and LI-COR Image Studio Acquisition Software (LI-COR Biosciences, NE). Mouse primary muscle fibroblasts were used as the positive control for GM130.
Iijima H., Wang K., D’Amico E., Tang W.Y., Rogers R.J., Jakicic J.M, & Ambrosio F. (2023). Exercise-primed extracellular vesicles improve cell-matrix adhesion and chondrocyte health. Research Square.
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8

Extracellular Vesicle Protein Analysis

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sEV and cells were resuspended in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.25% deoxycholic acid, 1 % NP-40, 1 mM EDTA, pH 7.4, purchased from Millipore Sigma) supplemented with SDS 0.1% and protease and phosphatase inhibitors (Biotool), and incubated on ice for 20 min prior to centrifugation at 12,000 g for 10 min. Supernatants were used for western blotting in SDS-PAGE electrophoresis. sEV-SEC and NV-SEC samples were mixed with 10% RIPA buffer. Protein concentration was determined by a BCA kit (Thermofisher) in an Eon microplate controlled by Gen5 software (Biotek). The following antibodies were used: ALG-2-Interacting Protein X (ALIX) (ab186429, Abcam), TSG101 (sc-7964, Santa Cruz), CD9 (ab92726, Abcam), CD63 (ab68418), GM130 (sc-55590, Santa Cruz), Calnexin (CANX) (ab22595, Abcam), Alyref (also named THOC4, 12655 Cell Signaling), Fus (4885, Cell Signaling), Vinculin (MAB3574, Millipore-Sigma), goat anti-rabbit IgG (H+L)-HRP conjugate secondary antibody (1706515, Bio-Rad) and sheep anti-mouse- HRP conjugate (NA931V, Amersham). All antibodies from abcam, Cell Signaling and Millipore-Sigma were used at a 1/1000 dilution, while those from Santa Cruz were at 1/250. Anti-rabbit and anti-mouse secondary antibodies were used at a 1/5,000 and 1/3,000 dilution, respectively.
Garcia-Martin R., Wang G., Brandão B.B., Zanotto T.M., Shah S., Patel S.K., Schilling B, & Kahn C.R. (2021). MicroRNA sequence codes for small extracellular vesicle release and cellular retention. Nature, 601(7893), 446-451.
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9

Comprehensive Antibody Characterization Panel

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We purchased antibodies against Ki67 (Clone: MM1), Villin (Santa Cruz, sc-7672), Gapdh (Clone: 6C5), Calnexin (Clone: 37), GM130 (Clone: 35), Adenine nucleotide translocase (Santa Cruz, sc-9299), Lyn (Santa Cruz, sc-15), GP130 (Santa Cruz, sc-9045), Tubulin (Clone: B-5-1-2), EpCAM-PE (Clone: 9C4), CD24-APC (Clone: ML5), CD45-PE-Cy5 (Clone: 30-F11), and PDI (Clone, 1D3). We generated the antibody against mouse ZIP7 by immunizing a rabbit with a peptide corresponding to the GNTGPRDGPVKPQSPEE sequence of mouse ZIP7, which was affinity-purified by using the antigen peptide.
Ohashi W., Kimura S., Iwanaga T., Furusawa Y., Irié T., Izumi H., Watanabe T., Hijikata A., Hara T., Ohara O., Koseki H., Sato T., Robine S., Mori H., Hattori Y., Watarai H., Mishima K., Ohno H., Hase K, & Fukada T. (2016). Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress. PLoS Genetics, 12(10), e1006349.
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10

EV Protein Characterization by Western Blot

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Samples were lysed in RIPA (radioimmunoprecipitation assay) buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl [pH 8.0]) for 15 min at 4°C. Total protein amounts of EVs and other secretome samples were estimated using a micro BCA protein assay reagent kit (Thermo Fisher Scientific) in accordance with the manufacturer’s specifications. Proteins were separated on SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane, and probed with respective antibodies: CD9 (Santa Cruz, sc-13118), TSG101 (Santa Cruz, sc-6037), CD63 (Santa Cruz, sc-15363), ALPPL2 (Abcam, ab54780), calnexin (Santa Cruz, sc-23954), GM-130 (Santa Cruz, sc-55591), cytochrome c (Santa Cruz, sc-13156), nucleoporin p62 (Santa Cruz, sc-48393), and secondary anti-mouse immunoglobulin HRP (Santa Cruz, sc-516102) according to standard protocols. Protein bands were detected on an X-ray film using clarity western ECL substrate (Bio-Rad).
Shin H.S., Jung S.B., Park S., Dua P, & Lee D.K. (2019). ALPPL2 Is a Potential Diagnostic Biomarker for Pancreatic Cancer-Derived Extracellular Vesicles. Molecular Therapy. Methods & Clinical Development, 15, 204-210.
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