Computational protocol: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation

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[…] Cells were washed once with PBS and either directly fixed with 3.7% formaldehyde or first incubated in pre-extraction buffer (20 mM HEPES pH 7.4, 110 mM KOAc, 2 mM Mg(OAc)2, 1 mM EDTA, 2 mM DTT, 0.2% Triton X-100) for 1 min, followed by fixation with 3.7% formaldehyde for 10 min. After two washes with PBS, cells were stained with DNA stain Hoechst 33342 and membrane dye DiOC6 for 10 min, followed by brief wash with PBS, and mounted in Fluoromount G (Southern Biotech).Images were obtained using a Nikon TE-2000 microscope equipped with a 1.45 NA 100x objective, Sedat quad filter set, PIFOC Z-axis focus drive (Physik Instruments), and CoolSnapHQ High Speed Monochrome CCD camera (Photometrics) run by Metamorph image acquisition software. Image stacks (0.2 μm steps) were deconvolved using AutoquantX. Micrographs were saved from source programs as. tif files and prepared for figures using GIMP. [...] Sequence alignments were performed using ClustalW2 and transmembrane domains were predicted with TMHMM v2.0. ER retrieval sequence was detected using PSort II. […]

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