rabbit anti mouse anti eea 1 primary antibody  (Cell Signaling Technology Inc)

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Figure Lengend Snippet: (A) 2′-O-methyl modifications were introduced in all uracil residues of the miR-29b reverse strand as indicated. RAW264.7 cells were plated four hours before stimulation with DOTAP-embedded miR-29b, 2′-O-Me-modified miR-29b, or the control miR-127 (750 nM working concentration). TNFa was quantified in supernatants eighteen hours later. 2′-O-Me modifications were introduced in the miR-29b reverse strand before annealing to the unmodified guide strand. Results are represented as individual values of cytokine concentrations (pg/ml). Data from one representative experiment out of three is shown. * P <0.05 (Mann-Whitney). (B) Intracellular distribution of Alexa-488-labelled miR-29b 1hour after transfection of RAW264.7 cells was observed with a confocal fluorescence microscope. Top row: IF EEA-1 on fixed cells Bottom row: lysotracker in living cells. Scale bar = 20 µm for fluorescence images and overlays with differential interference contrast (DIC) (a–d, f–i) except for enlarged single cell images scale bar = 5 µm (e, j). (C) Chloroquine (CQ) was added to the plated RAW264.7 cells, at a final concentration of 10 nM, 30 minutes before stimulation with miR-29b or the miR-127 control (750 nM). Supernatants were harvested eighteen hours later for TNFa quantification. Results are represented as individual values of cytokine concentrations (pg/ml) compiled from two independent experiments. ** P <0.01 (Mann-Whitney) (D) RAW264.7 cells were stimulated with miR-29b, miR-127 (750 nM), the positive controls TLR-7-ligand imiquimod and R848, or were left untreated (NT), and were cultured eighteen hours with or without the TLR-7 antagonist IRS661. TNFa was quantified in supernatants. Results are presented as mean cytokine concentration of replicates (pg/ml) ± SEM. Data from one representative experiment out of three is shown.

Article Snippet: Cells were fixed with 4% paraformaldehyde, permeabilized with 0,2% Tween20, and incubated with 5% goat blocking serum in PBS and incubated with 1 µg/ml primary rabbit anti- mouse EEA-1 antibodies (Abcam, Paris, France) for 1 hour at 37°C followed by incubation with 8 µg/ml secondary goat anti-rabbit ALEXA-555-conjugated antibody for 45 min. at room temperature.

Techniques: Modification, Concentration Assay, MANN-WHITNEY, Transfection, Fluorescence, Microscopy, Cell Culture