Sc 25778

Manufactured by Merck Group

Sc-25778 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of Sc-25778 is to provide a reliable and consistent tool for researchers and scientists to conduct their experiments and analyses.

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4 protocols using sc 25778

Detecting LC3 and p62 in Brain Tissue

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For the detection of LC3 in brain tissue, a membrane fraction enriched in autophagosomes was prepared as described previously (Chu et al, 2009), and for p62 after extracting soluble protein as in Wang et al (2007); 15 μg total protein was separated on 14% SDS–PAGE using a Laemmli buffer system. Proteins of interest were detected after transfer to PVDF membranes using primary antibodies NB100‐2220 (LC3, Novus Biologicals), P0067 (p62, Sigma), sc‐25778, and sc‐11397 (GAPDH and calnexin, respectively, Santa Cruz), all at a 1:1,000 dilution.

Nitschke F., Sullivan M.A., Wang P., Zhao X., Chown E.E., Perri A.M., Israelian L., Juana‐López L., Bovolenta P., Rodríguez de Córdoba S., Steup M, & Minassian B.A. (2017). Abnormal glycogen chain length pattern, not hyperphosphorylation, is critical in Lafora disease. EMBO Molecular Medicine, 9(7), 906-917.

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Protein Extraction and Western Blot Analysis

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Total proteins from different organs or from adipocytes were extracted in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, pH 8.0, 50 mM NaF, 1 mM sodium glycerophosphate, 5 mM pyrophosphate, 0.27 M sucrose, 1% Triton X-100, 0.1 mM PMSF, 0.1% 2-mercaptoethanol, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) and centrifuged at 8,600 × g for 20 min at 4°C. Extracts were separated by SDS-PAGE and transferred to 0.2-µm-pore-size nitrocellulose membranes (Bio-Rad). Blots were probed with primary antibodies to phospho-JNK (Thr183/Tyr185; 4668), total JNK (9252), phospho-AMPKα (Thr172; 2531), total AMPKα (2603), and phospho-p38 (Thr180/Tyr182; 9211; all from Cell Signaling Technology), p38 from Santa Cruz Biotechnology (sc-535), and GAPDH (sc-25778) and vinculin (V4505; Sigma-Aldrich). All antibodies were used at 1:1,000. After washes, membranes were incubated with an appropriate horseradish peroxidase–conjugated secondary antibody (GE Healthcare), and signals were detected using an enhanced chemiluminescent substrate (Clarity Western ECL substrate; Bio-Rad). Protein levels were analyzed by optical density measured with ImageJ (National Institutes of Health). Results were normalized to the not phosphorylated form.

Manieri E., Herrera-Melle L., Mora A., Tomás-Loba A., Leiva-Vega L., Fernández D.I., Rodríguez E., Morán L., Hernández-Cosido L., Torres J.L., Seoane L.M., Cubero F.J., Marcos M, & Sabio G. (2019). Adiponectin accounts for gender differences in hepatocellular carcinoma incidence. The Journal of Experimental Medicine, 216(5), 1108-1119.

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Immunoblotting Assay for B16F10 Cells

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For immunoblotting, B16F10 cells were lysed by PRO-PREP protein extraction solution (iNtRON Biotechnology). Briefly, 5 × 10⁶ cells were immersed in 400 μl of the PRO-PREP solution and homogenized in ice for 10–20 min. The mixture of cell lysates was then centrifuged at 13,000 rpm for 15 min. The supernatant was collected, and the protein concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The polypeptides were separated on 4–20% SDS–PAGE gradient gels and then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Ras Laboratories). The member was blocked with 5% skim milk (Difco Laboratories) and underwent overnight antibody incubation Primary Abs against the following proteins were used: human/mouse NLRP3 (1:1000, AP-20B-0014, AdipoGen), human NCOA6 (1:1000, A300-411A, Bethyl Laboratories), human/mouse ASC (1:1000, sc-514414, Santa Cruz Biotechnology; AG-25B-0006, AdipoGen), IL-1β (1:500, AF-401-NA, R&D Systems), mouse cleaved caspase-1 (1:1000, AG-20B-0042, AdipoGen), FLAG (1:1000, sc-166355, Santa Cruz Biotechnology), Myc (1:1000, ab13836, Abcam), GAPDH (1:1000, sc-25778, Sigma).

Li D., Wang X., Park D.J., Lee D.H, & Oh S. (2023). Inhibitory Effects of Latilactobacillus curvatus BYB3 Cell-Free Extract on Human Melanoma B16F10 Cells and Tumorigenic Mice. Journal of Microbiology and Biotechnology, 34(3), 589-595.

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Evaluation of Cholesterol Regulation Proteins

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The assay was adapted from^{29 (link)}. HeLa WT and NPC1 KO cells were transfected with the indicated siRNAs (Silencer Select, Thermo Fisher) for 72 h, then incubated for 16 h in either full medium (DMEM + 10% FCS + 1% Pen-Strep) or cholesterol-depletion medium (DMEM + 5% LPDS + 10 µM mevalonate + 1 µM atorvastatin (Sigma)), respectively. Then, 50 µg/ml LDL was added to the respective samples for 2–24 h at 37 °C. 25 µg/ml calpain inhibitor I (Cayman via Biomol) was added for 1 h at 37 °C before harvesting the cells in PBS + complete protease inhibitor cocktail (Roche). Cells were lysed in PBS + complete protease inhibitor cocktail containing 0.5% Triton-X 100. The lysates were analyzed via SDS-PAGE followed by Western blotting and subsequent immunodetection using anti-SREBP2 (R&D Systems, MAB7119, 1:250 in 5% skim milk), anti-LDLR (abcam, ab52818, 1:1000 in 5% skim milk), anti-HMGCR (abcam, ab1774830, 1:1000 in 5% skim milk), anti-LIMP-2 (self-made, 1:2000 in 5% skim milk), anti-NPC1 (Novus Biologicals, NB400-148, 1:1000 in 5% skim milk), anti-GAPDH (Santa Cruz Biotechnology #sc-25778, 1:5000, 5% skim milk) and anti-Actin (Sigma, A2066, 1:8000 in 5% skim milk) antibodies.

Heybrock S., Kanerva K., Meng Y., Ing C., Liang A., Xiong Z.J., Weng X., Ah Kim Y., Collins R., Trimble W., Pomès R., Privé G.G., Annaert W., Schwake M., Heeren J., Lüllmann-Rauch R., Grinstein S., Ikonen E., Saftig P, & Neculai D. (2019). Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) is involved in lysosomal cholesterol export. Nature Communications, 10, 3521.

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