rabbit polyclonal antibody to md2  (Novus Biologicals)

Journal: Endocrinology

Article Title: Functional toll-like receptor 4 conferring lipopolysaccharide responsiveness is expressed in thyroid cells.

doi: 10.1210/en.2008-0345

Figure Lengend Snippet: FIG. 1. Expression of TLR4 and the accessory molecules (CD14 and MD2) in thyroid cells. Basal FRTL-5 and PCCL3 thyroid cells and whole rat thyroid tissue were assayed for expression of TLR4 and accessory molecules at mRNA and protein levels. A, Representative RT-PCR for TLR4, CD14, and MD2. Total RNA was extracted from starved FRTL-5 and PCCL3 thyroid cell lines and whole rat thyroid tissue. Spleen was run in parallel as a positive control. B, Representative Western blot of whole proteins obtained from FRTL-5 and PCCL3 cells at basal state and after TSH treatment (0.5 mIU/ml, 48 h). The same blots were reprobed with an anti--actin antibody to assess equal loading. In the lowest panel, NIS expression is shown to ensure effective TSH response of the cells.

Article Snippet: Rabbit polyclonal anti-MD2 antibody was from Abcam, Inc. (Cambridge, MA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Western Blot

Journal: Endocrinology

Article Title: Functional toll-like receptor 4 conferring lipopolysaccharide responsiveness is expressed in thyroid cells.

doi: 10.1210/en.2008-0345

Figure Lengend Snippet: FIG. 2. TLR4 and accessory molecules are localized at the plasma membrane in the thyroid cell. A, Nonpermeabilized FRTL-5 cells were analyzed for TLR4, CD14, and MD2 expression by flow cytometry under basal or TSH (0.5 mIU/ml, 48 h) conditions. Negative control represents the samples only labeled with the secondary FITC-conjugated antibody. The histogram in each panel plots the number of cells (events) on the vertical axis against the fluorescence intensity of the labeled antibody bound to the indicated protein on the horizontal axis. The horizontal line on each histogram indicates the fluorescence range that contains the indicated percentage of positive cells for the assayed protein in the population. Each histogram is representative of three independent experiments. B, Immunohistochemical staining for the LPS-receptor TLR4 and the accessory molecules CD14 and MD2 in normal whole rat thyroid tissue. Evident expression with a basolateral localization in the follicular cells is observed for the three molecules analyzed (magnification, 400). Insets display a particular region of the same figure (magnification, 1000). Negative control (NC) shows the absence of nondesired staining when specific antibodies were replaced by a nonrelated IgG from the same species.

Article Snippet: Rabbit polyclonal anti-MD2 antibody was from Abcam, Inc. (Cambridge, MA).

Techniques: Clinical Proteomics, Membrane, Expressing, Flow Cytometry, Negative Control, Labeling, Fluorescence, Immunohistochemical staining, Staining

Journal: Endocrinology

Article Title: Functional toll-like receptor 4 conferring lipopolysaccharide responsiveness is expressed in thyroid cells.

doi: 10.1210/en.2008-0345

Figure Lengend Snippet: FIG. 3. LPS binds to the thyroid cell plasma membrane. A, Nonpermeabilized basal FRTL-5 cells were assayed for LPS binding using FITC-LPS as probe. Cells were incubated for 1 h with 0.01–10 g/ml probe (f), probe plus an excess (500) of unlabeled LPS (Œ), or probe plus 10 g/ml anti-TLR4 blocking antibody (●), and analyzed by flow cytometry. Results were normalized in relation to the fluorescence intensity of unlabeled cells and expressed as mean SD of three independent experiments. B, Lysates from TSH-starved FRTL-5 cells treated or untreated as indicated for 30 min were immunoprecipitated (IP) with either control goat IgG or anti-TLR4 antibody. The bound proteins were detected by Western blot with anti-CD14 or anti-MD2 antibodies. Lower panel, Whole cell extracts (WCE) were also subjected directly to Western blot analysis with the same antibody used for immunoprecipitation to show that equal amounts of TLR4 were expressed at the starting point. FIG. 4. LPS increases TSH-stimulated iodide uptake and NIS protein expression in thyroid cells. Basal FRTL-5 and PCCL3 cells were treated with LPS alone (100 ng/ml), TSH alone (0.5 mIU/ml), or TSH plus LPS (10 and 100 ng/ml) for 48 h. A, Iodide uptake level in basal, LPS, TSH, and TSH plus LPS treated cells. FRTL-5 cells are shown in black bars and PCCL3 in gray bars. Each value represents the mean SD of pmol I/g DNA of three independent experiments done in triplicate. *, P 0.01 and **, P 0.001 vs. TSH alone; #, P 0.005 vs. basal cells (Student-Newman-Keuls multiple comparisons test). B, LPS-induced increase in the TSH-stimulated iodide uptake varies with the time of incubation. Basal FRTL-5 cells were treated as previously described for different times (24–72 h). Each value represents the mean SD of pmol I/g DNA of three independent experiments done in triplicate. *, P 0.05 and **, P 0.001 vs. TSH alone; #, P 0.001 vs. basal cells (Student-Newman-Keuls multiple comparisons test). C, Representative Western blot analysis of whole cell extract assayed for NIS expression. -Actin, a nonrelated thyroid housekeeping gene, was used to correct loading differences. Densitometric analysis was performed to determine the relative increase (fold increase) of NIS normalized to - actin. The value of TSH alone was set up arbitrarily as 1.0. The results represent the mean of three independent experiments.

Article Snippet: Rabbit polyclonal anti-MD2 antibody was from Abcam, Inc. (Cambridge, MA).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Incubation, Blocking Assay, Flow Cytometry, Fluorescence, Immunoprecipitation, Control, Western Blot, Expressing