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Golgin 97

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Golgin-97 is a protein that is localized to the Golgi apparatus in eukaryotic cells. It is involved in the organization and structural maintenance of the Golgi complex. Golgin-97 plays a role in the tethering and fusion of transport vesicles to the Golgi membrane.

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Lab products found in correlation

Rhodamine phalloidin, by Thermo Fisher Scientific (2 mentions) Gm130, by BD (2 mentions) γ tubulin, by Abcam (2 mentions) Gorab, by Proteintech (2 mentions) Acetylated α tubulin, by Merck Group (2 mentions) Krt17, by Abcam (2 mentions) Lectin helix pomatiaagglutinin hpa, by Thermo Fisher Scientific (2 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Krt14, by Fortrea (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions) Deadend fluorometric tunel system, by Promega (2 mentions) Ds qi1mc camera, by Nikon (2 mentions) Huc d, by Thermo Fisher Scientific (1 mentions) β catenin, by Merck Group (1 mentions) Strep tag, by IBA Lifesciences (1 mentions) N cadherin, by Thermo Fisher Scientific (1 mentions) β catenin, by Cell Signaling Technology (1 mentions) Rela p65, by Cell Signaling Technology (1 mentions) Vamp3, by Synaptic Systems (1 mentions) Syntaxin5, by Synaptic Systems (1 mentions)

10 protocols using golgin 97

1

Immunofluorescence Antibody Staining Protocols

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The following antibodies and reagents were used in these studies: Mouse antibodies against HuC/D (#A21271, 1:500; Molecular Probes), β-catenin (#C7207, 1:200; Sigma), β-catenin (#9562, 1:500; Cell Signaling Technologies), Golgin97 (#A21270, 1:100; Invitrogen), Strep-tag (#2-1507-001, 1:500; IBA Lifesciences), CLIMP63 (#C5840-93, 1:1,000; United States Biological), aPKCζ/λ (#SC-17781, 1:200; Santa Cruz Biotechnology), GM130 (#610822, 1:500; BD Biosciences), rat antibodies against N-cadherin (#13-2100, 1:200; Invitrogen), rabbit antisera against Sox2 (#48-1400, 1:500; Invitrogen), calreticulin (#ab2907, 1:1,000; Abcam), and the chemicals rhodamine-phalloidin (#R415, 1:250; Invitrogen).
Herrera A., Menendez A., Torroba B., Ochoa A, & Pons S. (2021). Dbnl and β-catenin promote pro-N-cadherin processing to maintain apico-basal polarity. The Journal of Cell Biology, 220(6), e202007055.
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2

Antibody Sources for Protein Detection

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Rabbit polyclonal antibodies against Syntaxin-5, VAMP3, and VAMP8 were obtained from Synaptic Systems; rabbit polyclonal against β-actin, p65RelA, and mTOR from Cell Signaling; rabbit polyclonal anti-c-Jun from Santa Cruz Biotechnology; mouse monoclonal anti-SHP-1 from Abcam; mouse monoclonal antibody anti-NLRP3 from AdipoGen Life Sciences; mouse monoclonal antibodies against Calnexin and Golgin 97 from Invitrogen. The rabbit anti-Syt XI polyclonal antibody [59 (link)] was kindly provided by Dr Mitsunori Fukuda (Tohoku University). The rabbit polyclonal antibodies anti-PTP-PEST 2530 [60 (link)] were kindly provided by Dr Michel L. Tremblay (McGill University). The secondary HRP-conjugated antibodies anti-mouse and anti-rabbit were obtained from Sigma Aldrich. The secondary antibody anti-rabbit conjugated to AlexaFluor 488 and the secondary antibody goat anti-mouse conjugated to AlexaFluor 568 used for immunofluorescence were from Invitrogen-Molecular probes.
Guay-Vincent M.M., Matte C., Berthiaume A.M., Olivier M., Jaramillo M, & Descoteaux A. (2022). Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates. PLoS Pathogens, 18(10), e1010640.
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3

Immunofluorescence Labeling of Tissue Specimens

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Immunofluorescence labeling of tissue specimens was performed as
described previously (Dai et al., 2013 (link)).
Cells were fixed in 4% PFA and blocked in 1% BSA prior to
incubating with primary antibodies. The following primary antibodies were used:
GORAB, 1:500 (Proteintech); KRT14, 1:1,000 (Covance); KRT1, 1:2000 (Roop et al.,
1987); KRT17, 1:200 (Abcam); LOR, 1:500 (Covance); NGFR, 1:200 (Promega); LEF1,
1:100 (Cell Signaling, Danvers, MA); acetylated α-tubulin, 1:600
(Sigma); γ-tubulin, 1:500 (Abcam); ARL13B, 1:100
(#73–287, NeuroMab); lectin Helix pomatiaagglutinin (HPA), 1:1,000 (Invitrogen); Golgin97, 1:1,000 (Molecular Probes),
and Ki67, 1:1000 (BD Pharmingen). AlexaFluor-conjugated secondary antibodies
(1:250) were from Life Technologies. Sections were sealed in mounting medium
with DAPI (Vector Laboratories). TUNEL staining were performed with DeadEnd
Fluorometric TUNEL System (Promega). Images were acquired by Nikon
80i fitted with Nikon DS-Qi1Mc camera and processed with
Photoshop 5.5 CS.
Liu Y., Snedecor E.R., Choi Y.J., Yang N., Zhang X., Xu Y., Han Y., Jones E.C., Shroyer K.R., Clark R.A., Zhang L., Qin C, & Chen J. (2015). Gorab is required for dermal condensate cells to respond to hedgehog signals during hair follicle morphogenesis. The Journal of investigative dermatology, 136(2), 378-386.
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4

Immunofluorescence Labeling of Tissue Specimens

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Immunofluorescence labeling of tissue specimens was performed as
described previously (Dai et al., 2013 (link)).
Cells were fixed in 4% PFA and blocked in 1% BSA prior to
incubating with primary antibodies. The following primary antibodies were used:
GORAB, 1:500 (Proteintech); KRT14, 1:1,000 (Covance); KRT1, 1:2000 (Roop et al.,
1987); KRT17, 1:200 (Abcam); LOR, 1:500 (Covance); NGFR, 1:200 (Promega); LEF1,
1:100 (Cell Signaling, Danvers, MA); acetylated α-tubulin, 1:600
(Sigma); γ-tubulin, 1:500 (Abcam); ARL13B, 1:100
(#73–287, NeuroMab); lectin Helix pomatiaagglutinin (HPA), 1:1,000 (Invitrogen); Golgin97, 1:1,000 (Molecular Probes),
and Ki67, 1:1000 (BD Pharmingen). AlexaFluor-conjugated secondary antibodies
(1:250) were from Life Technologies. Sections were sealed in mounting medium
with DAPI (Vector Laboratories). TUNEL staining were performed with DeadEnd
Fluorometric TUNEL System (Promega). Images were acquired by Nikon
80i fitted with Nikon DS-Qi1Mc camera and processed with
Photoshop 5.5 CS.
Liu Y., Snedecor E.R., Choi Y.J., Yang N., Zhang X., Xu Y., Han Y., Jones E.C., Shroyer K.R., Clark R.A., Zhang L., Qin C, & Chen J. (2015). Gorab is required for dermal condensate cells to respond to hedgehog signals during hair follicle morphogenesis. The Journal of investigative dermatology, 136(2), 378-386.
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5

Validating Golgin Antibody Specificity

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Commercial antibodies used were GM130 (BD Biosciences 610823), golgin160 (Sigma [St. Louis, MO, USA] HPA039809), GMAP210 (Sigma HPA070684), golgin97 (Thermo Fisher Scientific A‐21270), golgin245 (BD Biosciences [San Jose, CA, USA] 611281), GCC88 (Sigma HPA019369), GCC185 (Sigma HPA035849), and Flag (Sigma F1804, F7425). All antibodies were validated for their specificity by confocal microscopy using the expression of constructs of the golgin in question (listed above) fused to a Flag tag.
Ziltener P., Rebane A.A., Graham M., Ernst A.M, & Rothman J.E. (2020). The golgin family exhibits a propensity to form condensates in living cells. Febs Letters, 594(19), 3086-3094.
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6

Immunofluorescence Imaging of B-ALL Cells

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Human B-ALL cells were seeded at a concentration 5 × 105 cells/mL to a poly-L-lysine (Sigma-Aldrich)-coated 8-well removable chamber microscope slide (ibidi) and allowed to adhere for 24 hours at 37°C prior to treatment. At harvest, the wells were washed twice with cold PBS, fixed with 4.2% paraformaldehyde (PMA) at 4°C for 10 minutes, permeabilized with 0.2% Triton X-100 (Sigma Aldrich) for 10 minutes at room temperature, and blocked for 30 minutes at room temperature. Wells were stained for Sig15 (Invitrogen; 1:400) and Golgin-97 (Thermo Fisher Scientific; Clone CDF4; 1:200) for 20 minutes at room temperature, washed in triplicate with PBS, and stained with fluorescent secondary antibodies against either antigen. Slides were mounted using Prolong Gold Antifade with DAPI (Invitrogen) and imaged on an Olympus FV1000 confocal microscope. All colocalization analysis was performed using the Coloc2 plugin in Fiji (23 (link)).
Pillsbury C.E., Dougan J., Rabe J.L., Fonseca J.A., Zhou C., Evans A.N., Abukharma H., Ichoku O., Gonzalez-Flamenco G., Park S.I., Aljudi A., DeRyckere D., Castellino S.M., Rafiq S., Langermann S., Liu L.N., Henry C.J, & Porter C.C. (2023). Siglec-15 Promotes Evasion of Adaptive Immunity in B-cell Acute Lymphoblastic Leukemia. Cancer Research Communications, 3(7), 1248-1259.
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7

Subcellular Localization of Slc16a13 in HEK Cells

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HEK-Slc16a13-FLAG or HEK-control cells were seeded onto poly-D-lysine coated cover glasses, fixed in 4% PFA, blocked with 10% goat normal serum in PBS, and incubated with the following antibodies and dyes: FLAG (ThermoFisher Scientific, PA1-984B, 1:500), Golgin-97 (ThermoFisher Scientific, A-21270, 1:100), Calnexin (Novus Biologicals, NB300-518, 1:100), goat anti-rabbit Alexa Fluor 488 secondary antibody (ThermoFisher Scientific, A-11008, 1:500), goat anti-mouse Alexa Fluor 594 (ThermoFisher Scientific, R37121, 1:500), Alexa Fluor 594 wheat germ agglutinin (ThermoFisher Scientific, W11262, 1:100), MitoTracker CMXRos (ThermoFisher Scientific, M7512, 100 nM) and Hoechst 33342 (Invitrogen, 1:2000). Microscopy has been performed at the Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems).
Schumann T., König J., von Loeffelholz C., Vatner D.F., Zhang D., Perry R.J., Bernier M., Chami J., Henke C., Kurzbach A., El-Agroudy N.N., Willmes D.M., Pesta D., de Cabo R., O´Sullivan J.F., Simon E., Shulman G.I., Hamilton B.S, & Birkenfeld A.L. (2021). Deletion of the diabetes candidate gene Slc16a13 in mice attenuates diet-induced ectopic lipid accumulation and insulin resistance. Communications Biology, 4, 826.
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8

Engineered Breast Cancer Cell Lines

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MDA-MB-231 and Cal51 breast cancer cells were grown in DMEM supplemented with 10% (vol/vol) FBS, 2 mM L-glutamine, and 50 μg/mL penicillin/streptomycin at 37°C and 5% (Cal51) or 10% (MDA-MB-231) CO2. RFP-tagged histone H2B and GFP-tagged α-tubulin were stably integrated into MDA-MB-231 cells by transfection in a pBabe vector, and into Cal51 cells by lentiviral infection (Millipore LentiBrite™). Single clones were isolated with cloning cylinders (MDA-MB-231) or by single-cell sorting (Cal51). All paclitaxel and MG132 used in cell culture were dissolved in DMSO. Final concentration of DMSO in media was 0.1%. Chromosome spreads and immunofluorescence were performed as in (25 (link)). Staining was performed with antibodies to α-tubulin (YL½; Serotec) and Golgin-97 (Life Technologies).
Zasadil L.M., Andersen K.A., Yeum D., Rocque G.B., Wilke L.G., Tevaarwerk A.J., Raines R.T., Burkard M.E, & Weaver B.A. (2014). Cytotoxicity of paclitaxel in breast cancer is due to chromosome missegregation on multipolar spindles. Science translational medicine, 6(229), 229ra43.
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9

Immunofluorescence Localization of Organelle Markers

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Primary antibodies used included: protein disulfide isomerase (PDI, mouse anti‐human (Prod# MA3019); 1:250, Life Technologies), golgin‐97 (rabbit anti‐human (Prod #A21270); conc. 1:100, Life Technologies), and lysosomal‐associated membrane protein 1 (LAMP1, mouse anti‐human (ab25630); 1:100, Abcam Cambridge, MA). Secondary antibodies were: AlexaFluor488 (anti‐mouse (IgG1), conc. 1:750), AlexaFluor555 (anti‐mouse(IgG2a), conc. 1:750), and AlexaFluor647 (anti‐rabbit, conc. 1:750), all from Life Technologies.
Weidner J., Jarenbäck L., Åberg I., Westergren‐Thorsson G., Ankerst J., Bjermer L, & Tufvesson E. (2018). Endoplasmic reticulum, Golgi, and lysosomes are disorganized in lung fibroblasts from chronic obstructive pulmonary disease patients. Physiological Reports, 6(5), e13584.
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10

Immunofluorescence Staining of Dendritic Cells

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DC cultured in 8-well chamber slides (Nunc) were fixed (4 % paraformaldehyde), permeabilized (0.1 % Triton X-100), and stained with rhodamine phalloidin (Life Technologies) and DRAQ5 (eBioscience), to label F-actin and nuclei, respectively. The following primary antibodies were used for staining cells: AFP (AbD Serotec), CD206 (BioLegend), CD36, Tyrosinase, EEA-1, LAMP-1, KDEL, ERGIC-53 (all Santa Cruz Biotechnology), golgin-97 (Life Technologies), TGN46 (Sigma), mouse and rabbit IgG isotype controls (R&D Systems), and anti-Alexa Fluor 488 (to amplify the fluorescent signal of Alexa Fluor 488-labeled protein; Life Technologies). The following secondary antibodies were used: goat anti-mouse Alexa Fluor 488 and 555, goat anti-rabbit Alexa Fluor 488 and 555 (all from Cell Signaling Technology), donkey anti-goat Cy3 (Jackson ImmunoResearch), donkey anti-mouse Alexa Fluor-488, and donkey anti-rabbit Alexa Fluor-488 (both Life Technologies). Images were acquired using a Leica TCS-SL confocal microscope.
Pardee A.D., Yano H., Weinstein A.M., Ponce A.A., Ethridge A.D., Normolle D.P., Vujanovic L., Mizejewski G.J., Watkins S.C, & Butterfield L.H. (2015). Route of antigen delivery impacts the immunostimulatory activity of dendritic cell-based vaccines for hepatocellular carcinoma. Journal for Immunotherapy of Cancer, 3, 32.
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