Differentiated TR5TY6 cells induced for 0, 1, 3, and 6 days were homogenized in RIPA buffer supplemented with protease inhibitors (Roche) and cell extracts clarified by centrifugation at 10,000 × g for 10 min at 4 °C. Protein concentrations were quantified using the BCA method and subjected to SDS-PAGE. Proteins were resolved in 10 or 15% polyacrylamide gels and transferred onto 0.45 µm nitrocellulose membranes (Amersham). Blocking with 5% milk powder in PBS was followed by overnight incubation at 4 °C with the primary antibodies mouse anti-tyrosinase (1:2000, Thermo Fisher Scientific, Cat#MS-800-P1), rabbit anti-Lamp1 (1:250, GeneTex, Cat#GTX19294), rabbit anti-LC3 (1:1000, Novus Biologicals, Cat#NB100-2220), guinea pig anti-p62 (1:1000, Progen, Cat#GP62-C) or mouse anti-β-actin (1:5000, Sigma-Aldrich, Cat#A5441). Incubation with the secondary antibodies donkey anti-rabbit, sheep anti-mouse (both 1:5000, from Amersham), and goat anti-guinea pig (1:1000, Santa Cruz Biotechnology) was performed for 1 h at RT. Band densitometry, normalized to β-actin expression, were measured using ImageJ image analysis software.
Nb100 2220
Manufactured by Progen Biotechnik
The NB100-2220 is a laboratory centrifuge designed for general-purpose applications. It features a maximum speed of 22,000 rpm and can accommodate a variety of rotor types to suit different sample sizes and volumes. The centrifuge provides reliable and consistent performance for a wide range of centrifugation needs in research and clinical settings.
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Lab products found in correlation
Protease inhibitor, by Roche (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Gp62 c, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Gp62 c, by Abcam (1 mentions)
Map1lc3b, by Novus Biologicals (1 mentions)
Map1lc3b, by Nanotools (1 mentions)
2 protocols using nb100 2220
1
Quantitative Protein Analysis of Differentiated TR5TY6 Cells
2
Immunocytochemical Analyses of Autophagy
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Immunocytochemistry was performed as previously described (Bekbulat et al, 2020 (link)). Briefly, cells were grown on glass coverslips, fixated with 4% PFA, and permeabilized with 90% (v/v) methanol. Unspecific binding sites were blocked with 3% BSA in PBS followed by incubation with primary antibody, fluorophore‐conjugated secondary antibody, and DAPI. For sphingomyelin analyses, cells were treated with 500 nM BODIPY FL C5‐shingomyelin (Fisher, 11510306) in serum‐free EBSS for 1 h and thereafter were incubated for 1 h with bafilomycin A1 in EBSS. Cells were imaged using the laser scanning microscope LSM710 (Zeiss). Primary antibody: MAP1LC3B (Nanotools, 0260‐100/LC3‐2G6); MAP1LC3B (Novus, NB100‐2220); SQSTM1 (Progen, GP62‐C); LAMP2 (Abcam, ab25631); TGN46 (BioRad, AHP500); HA (Sigma, H6908). Secondary antibody: Cy3 anti‐mouse (ImmunoResearch, 715‐165‐151), Cy3 anti‐rabbit (ImmunoResearch, 711‐165‐152), Cy3 anti‐sheep (ImmunoResearch, 713‐165‐147), Cy5 anti‐mouse (ImmunoResearch, 715‐175‐150), Cy5 anti‐rabbit (ImmunoResearch, 711‐175‐152).
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