Lysopure nuclear and cytoplasmic extractor kit

Following stimulation, fibroblasts were lysed with RIPA buffer (Cell Signaling Technology) containing phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA). Nuclear lysates were prepared using a LysoPure™ Nuclear and Cytoplasmic Extractor Kit (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) containing protease inhibitor cocktail Set III (FUJIFILM Wako Pure Chemical Corporation). Protein levels were quantified using precision red advanced protein assay reagent #2 (Cytoskeleton, Denver, CO, USA). Equal amounts of total protein were separated on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred onto an immobilon-P transfer membrane (Millipore, Billerica, MA, USA) by a PoweredBLOT 2M system (ATTO, Tokyo, Japan). The membranes were blocked with ImmunoBlock (KAC, Hyogo, Japan) for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. The membranes were washed with Tris-buffered saline with 0.1% Tween 20 and incubated with HRP-conjugated secondary antibodies. After being washed, the membranes were developed with Luminata Western HRP substrate (Millipore, Billerica, MA, USA), and the bands were detected with ImageQuant™ LAS 4000 (FUJIFILM Wako Pure Chemical Corporation). The intensity of the bands was quantified using ImageJ software (NIH, Bethesda, MD, USA).

LysoPure™ Nuclear and Cytoplasmic Extractor Kit (Wako; catalogue #295-73901) allowed us to extract cytoplasmic, soluble and insoluble nuclear fractions to elucidate the intracellular localization of domains of (P)RR. In accordance with the manufacturer’s instructions, the insoluble nuclear fraction was extracted using SDS lysis buffer and sonication after the extraction from cytoplasmic and soluble nuclear fractions.

After treating the cells, as described in the Figure legends, we lysed and separated them into subcellular fractions using the Lysopure Nuclear and Cytoplasmic Extractor kit (Wako Tokyo, Japan) according to the manufacturer’s protocol. Samples were separated by electrophoresis onto a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA) for transferring. The membrane was incubated in diluted primary antibody solution (1/500–1/1000) overnight (14–16 h) under agitation at 4°C. The PVDF membrane was incubated in horse radish peroxidase (HRP)-conjugated secondary antibody (1/10000 in TBST buffer) for 1 h. Signals were detected using a Clarity western ECL substrate (BioRad, Hercules, CA) and X-ray film (Carestream, NY, USA).

Tissue and cell samples were solubilized in RIPA buffer with freshly added protease inhibitors (Sigma, St. Louis, MO). 10–50 micrograms of protein was loaded on any kD™ Mini‐PROTEIN TGX gels (Hercules, CA) transferred to PVDF membranes, and analyzed by western blotting using the ECL method. Molecular weight was calibrated using Precision Plus Protein Dual Color Standards (Bio‐Rad). Nuclear/cytosolic fractions were isolated using Lysopure ™ Nuclear and Cytoplasmic Extractor Kit (WAKO Chemical, Osaka, Japan), according to the manufacturer's protocol. Protein levels were quantified by ImageJ software (National Institutes of Health, Bethesda, MD).