Journal:

Article Title: Asymmetric localization of flotillins/reggies in preassembled platforms confers inherent polarity to hematopoietic cells

doi: 10.1073/pnas.1331629100

Figure Lengend Snippet: Exclusive asymmetric localization of flotillins in PAPs and their DRM partitioning in resting cells. (a) Expression of flotillins in different hematopoietic cells. Lanes 1–9: pro-B cell line KM3, pre-B cell Nalm6, Epstein–Barr virus-transformed mature B cell lines Dakiki, Raji, plasma B cell line HSSultan, peripheral blood B lymphocytes, T lymphocytes, T cell leukemic Jurkat cells, and the promonocytic cell line, U937, respectively. M, marker lane. (A) PCR amplification of flotillin-1. GAPDH amplification serves as control. (B and C) Detection of flotillin-1 and -2 by Western blotting. Both flotillins run at 48 kDa under reducing conditions and the Ponceau stainings serve as loading controls. (b) Nonactivated pre-B cell line Nalm6 (A), mature B cell lines Raji (B), Ramos (C), T leukemic Jurkat cells (D), and promonocytic cell line U937 (E) were stained with mouse monoclonal antiflotillin-1 (green) costained with affinity-purified rabbit polyclonal anti-reggie-1/flotillin-2 (red) antibodies. All of the cells exhibited the cap-like staining. (c) Reggie-1-EGFP is selectively targeted to the PAPs. Jurkat cells were electroporated with the plasmid and the cells were analyzed by laser scanning confocal microscopy. The overexpressed fusion protein was also targeted to the PAPs. (d) Jurkat cells were stained with antiflotillin-1 antibodies and costained with phalloidin green. Intense intracellular staining of flotillins shows the outlines of nuclei (arrowheads) and helps perceive the orientation of the PAPs with respect to the nucleus. Bright-field images (differential interference contrast microscopy, DIC) show the intact, round morphology of these cells. (e) Detergent insolubility of flotillins. Cells (A–E as in b) were lysed in cold 1% Triton X-100 and a sucrose gradient was overlaid onto the lysate as mentioned in Materials and Methods. The detergent-insoluble fractions 2–5 were identified by CTx-B-HRP and anti-lyn antibody staining. Note that the B cell receptor (IgM) under nonactivating conditions was excluded from the detergent-insoluble fractions. (f) The first three rows represent nonactivated Jurkat T cells stained with flotillin-2/reggie-1 antibodies (red) and anti-lck, anti-CD59, and anti-CD71 antibodies (green). The fourth row shows anti-ezrin, radixin, and moesin (ERM) (red) and antiflotillin-2 (green) costaining in U937 promonocytic cells. The bottom row represents the B cell line Raji stained with anti-CD21 (red) and antiflotillin-2 (green) antibodies. None of the molecules except flotillins showed a polarized localization. (Bars = 5 μm.)

Article Snippet: Goat anti-human IgM conjugated with HRP was from Southern Biotechnology Associates.

Techniques: Expressing, Transformation Assay, Marker, Amplification, Western Blot, Staining, Affinity Purification, Plasmid Preparation, Confocal Microscopy, Microscopy