dephosphorylation during prolonged LPS stimulation of mammalian macrophage cells.Figure 1Reversal of miRNP activity and its restoration during the LPS treatment of RAW 264.7 cells. Schematic representation of the experiments where RAW 264.7 or PEC cells were treated with 1ng/ml of LPS for different time points before the RNA and protein content were analyzed (A) Relative fold repression for let-7a reporter in RAW 264.7 cells during LPS treatment (mean+/- s.e.m., n=3) (B). The schematic representation of the constructs used for luciferase assays are shown. Relative levels of pro-inflammatory cytokines TNF-ɑ and IL-6 mRNA, a target of let-7a miRNA, have been plotted against time of LPS stimulation, 18S rRNA was used for normalization (mean+/- s.e.m., n=2 (IL-6), n=3 (TNF-α)) (C). Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Ago2 was pulled down using anti-FLAG beads and the levels of Ago-associated let-7a miRNA were measured by qRT-PCR. Quantity of immunoprecipitated Ago2 was detected in western blot analysis with anti-HA antibody and used for normalization of amounts of miRNA detected by qRT-PCR. Levels of cellular let-7a were measured by Northern blot and U6 RNA was used as loading control. Relative levels of cellular and Ago2 bound let-7a were plotted (mean+/- s.e.m., n=3) (D). Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Phosphorylated Ago2 (pY-Ago2) level was measured using 4G10 antibody specific for phosphorylated tyrosine. Phosphorylated Ago2 (pY-Ago2) band intensities were normalized against total Ago2 detected with HA specific antibody that was also used for Ago2 pull down (E, upper panel). Relative 4G10 intensities were plotted from three independent experiments (mean+/- s.e.m) (E, lower panel). Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Primary cells (PEC) were isolated from BALB/c mice and endogenous Ago2 was immunoprecipitated with anti-Ago2 (eIF2C2) antibody and phospho tyrosine level of Ago2 was measured using 4G10 antibody. Relative intensities of phospho-Ago2 against total amount of immunoprecipitated Ago2 were quantified and mentioned below the lanes (mean+/- s.e.m., n=3) (F, upper panel). Similar experiments were done with FH-Ago1 and Ago3 and levels of Phosphotyrosine in immunoprecipitated FH-Ago1 and FH-Ago3 were measured in RAW 264.7 cells (F, lower panel). Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Knockdown of PP2A, MKP1, MKP3 or SHP1/2 was checked by western blot analysis done with lysates of cells treated with respective siRNAs. β-Actin was used as loading control (G). Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Luciferase based let-7a miRNA reporter assay was done in LPS-treated cells downregulated for specific phosphatases. Levels at 0 hrs time point were taken as units (mean +/- s.e.m., n=4) (H). Effect of knockdown of different phosphatases on miRNA-association of Ago2 in 24hrs LPS treated RAW 264.7 cells. Ago2 associated let-7a was estimated by qRT-PCR in LPS treated cells depleted for specific phosphatases. let-7a miRNA level was normalized against respective FH-Ago2 bands (mean+/- s.e.m., n=3) (I). Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Phospho Ago2 levels were measured in siCon and siPP2A transfected cells upon 24hrs of LPS treatment using Phospho Tyrosine specific 4G10 antibody. HA-Ago2 was detected with anti-HA antibody (J). Immunoprecipitated Ago2 content was used for normalization for both miRNA and mRNA levels in above mentioned experiments. Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Cellular miRNA levels (K, left panel mean +/- s.e.m., n=3) and Ago2 associated miRNAs content in cells treated with siCon or siPP2A were measured using qRT-PCR and plotted (K, right panel, mean+/- s.e.m., n=3). Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Ago2 associated IL-1β mRNA level was estimated in siCon and siPP2A treated cells which were stimulated with LPS for 24hrs (mean+/- s.e.m., n=3) (L).