Article Figures & Data
Figures
- Figure 1.
Bioinformatics at human REST locus (A) and predicted REST protein isoforms derived from alternative splicing (B). Related tracks were retrieved from the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway). REST gene boundary is more than doubled by an alternate last exon (E5) which partially overlaps in opposite direction with exon 5 of NOA1. REST promoter harbors a CpG island and exhibits cell-independent active transcription as indicated by the chromatin state segmentation and H3K27Ac tracks. Predicted ORFs of the full-length and alternatively spliced REST mRNAs were briefly shown by indicating the start (blue star) and stop (red star) codons, while major domains (RD1 and RD2, repression domain 1 and 2; NLS, nuclear localization signal; and zinc fingers 1–9) of the full-length REST protein were illustrated in parallel to their coding sequences. Splice variants expressed in multiple tissues or cell lines were bolded. Locations of the mRNA and protein fragments targeted by real-time PCR primer sets (P1–P4), RNAi (shRESTa and shRESTb), and antibodies mentioned in the text were indicated. Note that only the conventional promoter is shown and that the internal region of E4 is unconserved as indicated by the “100 vertebrate conservation” track, which supports our finding that partial skipping of E4 is common (Chen and Miller, 2013).
- Figure 2.
Immunofluorescence analysis of REST subcellular localization in different cells with two different antibodies. Immunocytochemistry (ICC) assays were performed with two anti-REST sc-25398 (Santa Cruz) and ab21635 (Abcam), which are, respectively, against N and C terminus of REST, for C6, RN46A, and COS7 cells. For each cell line, two wells of cells under the same experimental conditions were stained with sc-25398 and ab21635, respectively. Briefly, cells cultured on poly-D-lysine-coated coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and incubated with sc-25398 (1:100) or ab21635 (1:200), followed by incubation with a goat anti-rabbit secondary antibody conjugated with Alexa Fluor 568 (1:500, Invitrogen). Nuclei were stained with Hoechst-33342 (Thermo Scientific), and cells were mounted on glass slides. Confocal microscopy was performed using a Leica TCS SP5 Spectral Confocal Microscope. For each cell line, all experimental conditions were kept the same for the two antibodies. Regardless of the cell-types, ICC with sc-25398 yielded predominant localization of REST in nucleus, whereas ICC with ab21635 indicated predominant colocalization of REST with microtubule (or cytoskeleton), suggesting that REST isoforms with different subcellular localization might be differentially recognized by different antibodies.
- Figure 3.
WB of REST expression in HEK-293T cells with two different antibodies. Two aliquots (25 µg for each) of three different HEK293T protein samples (T1, T2, and T3), which were isolated simultaneously with RNA and DNA by TRIzol reagent (Invitrogen), along with a Kaleidoscope marker (Bio-Rad) in between, were loaded on a 7.5% PAGE-SDS gel, followed by electrophoresis and electrotranslocation onto an Immun-Blot PVDF membrane (Bio-Rad), which was then cut into two halves for incubation with sc-25398 (1:250) and ab21635 (1:500), respectively, and subsequent incubation with a goat anti-rabbit IgG (Sigma-Aldrich, 1:2500). IR signals were detected using the VisiGlo Select HRP Chemiluminescent Substrate kit (Amresco) with an ECL-based LAS-3000 image system (Fujifilm). Note that the two antibodies yielded totally different profiles of IR bands.
Tables
Antibody Without addendum With addendum IHC-00141, ab28018, sc-15118 IHC-00141 ab28018 sc-15118 ab202962 Sample (n) Total 171 171 7 of 171 49 of 171 35 of 171 Young 11 11 ? 10 10 Aged 77 77 ? 21 14 AD 72 72 ? 18 11 MCI 11 11 ? - - Result All presented IHC data All presented IHC data Data not shown Data not shown Data not shown
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