Cells and whole tissue were extracted into Tris lysis buffer with protease inhibitor (Roche), disrupted by sonication, and quantified for normalization using Pierce BCA protein assay kit (Thermo Scientific). Antibodies were used at manufacturer recommended concentrations and included: anti-IL-10RA (rabbit polyclonal), anti-Cldn2 (rabbit polyclonal), and anti–β-actin from Abcam; IL-10 (human), anti-acH3K9 (C5B11, rabbit monoclonal), anti-Stat3 (79D7, rabbit monoclonal), and anti-pStat3 (Y705, rabbit polyclonal) from Cell Signaling; and anti-IL10RA (rabbit polyclonal) from ThermoFisher. The Western blotting antibodies, peroxidase Goat Anti-Rabbit IgG and peroxidase Goat Anti-Mouse IgG, were purchased from Jackson Laboratories. Western blotting substrates, Pierce ECL and SuperSignal West Femto, were purchased from ThermoFisher.
To localize IL-10RA, T84 cells were exposed to butyrate or buffer control, fixed and processed for microscopy as described (16 (link)). Cells were localized with anti-IL-10RA followed by AlexaFluor 488 secondary Ab and counter-stained with AlexaFluor 546 (Invitrogen). Fluorescence images were obtained using an AxioCam MRc5 attached to an AxioImager A1 microscope (Zeiss, Oberkochen, Germany).
To localize IL-10RA, T84 cells were exposed to butyrate or buffer control, fixed and processed for microscopy as described (16 (link)). Cells were localized with anti-IL-10RA followed by AlexaFluor 488 secondary Ab and counter-stained with AlexaFluor 546 (Invitrogen). Fluorescence images were obtained using an AxioCam MRc5 attached to an AxioImager A1 microscope (Zeiss, Oberkochen, Germany).
