Notably, the phagocytic capacity of monocyte-derived macrophages from patients with ITP were substantially reduced after priming with ivermectin

Notably, the phagocytic capacity of monocyte-derived macrophages from patients with ITP were substantially reduced after priming with ivermectin. ITP was associated with the underexpression of ADAP in splenic macrophages. Furthermore, macrophages from mice. Together, our data illustrate the ADAP-STAT1 module as a key regulatory component for platelet phagocytosis by macrophages and thus provide a potential therapeutic target for ITP. Materials and methods Clinical specimens Spleen tissues were collected from 10 patients with ITP who underwent splenectomy (3 females and 7 males, age range 13C71 years, median platelet counts 37.5??109/L) and 15 non-ITP patients with splenic trauma (5 females and 10 males, age range 17C72 years, median platelet counts 207??109/L). Formalin-fixed and paraffin-embedded tissue slides were prepared using the same standardization process. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood provided by 5 patients with ITP (3 females and 2 males, age range 25C71 years, median platelet counts 29??109/L). Patient characteristics were given in Suppl. Table?1. This study was approved by the Institutional Review Table of the Dushu Lake Hospital Affiliated to Soochow University or college and conducted in compliance with the Declaration of Helsinki. Written informed consent for the use of samples was acquired from all patients. Animals and were designed using the GPP sgRNA Designer [40] and cloned into a CRISPRCCas9-based lentiviral vector (plentiCRISPRv2, Addgene, 52962) as explained in [41]. Cloned oligonucleotides were as follows: sgRNA-luciferase activity following the manufacturers instructions (Promega). Expression and purification of recombinant proteins Human importin 5 (aa 400C538) was cloned into the pGEX-4T1 GST fusion vector (Cytiva) and expressed by BL21 (DE3) MGC102953 cells. GST-tagged proteins were purified with BeyoGold? GST-tag Purification Resin (Beyotime) and eluted with 10 mM L-glutathione, followed by concentration with an Amicon Ultra15 filter (30-kD cutoff, Sigma). The synthesized ADAP NLS1 peptide (KEREKKREKEEKKRLELEKKEQKEKEKKEQEIKKK), ADAP NLS2 peptide (KAKTEEKDLKKLKKQEKEEKDFRKKFK), and STAT1 NLS PI-103 peptide (HLQLKEQKNA) were purchased from Sangon. Immunoblotting and Immunoprecipitation Cells were lysed in lysis buffer (1% Triton X-100 (v/v) in 20?mM Tris-HCl (pH 8.3), 150?mM NaCl) supplemented with EDTA-free protease inhibitor cocktail (Roche). For immunoprecipitation experiments, cell lysates were precleared with protein G Sepharose beads (GE Healthcare) followed by incubation with antibodies at 4?C overnight. Immunoprecipitates were resolved by SDSCPAGE, and immunoblotting was performed with the indicated main antibodies. Immunoblots were developed with HRP- or IRDye-conjugated secondary antibodies and visualized by enhanced chemiluminescence or an Odyssey imaging system, respectively. For the GST pull-down assay, GST-importin 5 (aa 400C538) was incubated with cell lysates at 4?C for 1?h, followed by coupling to GST-tagged purification resin overnight at 4?C. Beads were then washed with lysis buffer and eluted in 1??Laemmli sample buffer before being subjected to immunoblotting analysis. Subcellular fractionation Cytoplasmic and nuclear fractionation was performed using subcellular lysis buffer (250?mM sucrose, 20?mM HEPES (pH 7.4), 10?mM KCl, 150?mM MgCl2, 1?mM EDTA, 1?mM EGTA) or nuclear lysis buffer (1% NP-40 (v/v) in 50?mM Tris-HCl (pH 8.3), 150?mM NaCl, 0.5% sodium deoxycholate, PI-103 0.1% SDS, 10% glycerol) supplemented with protease inhibitor cocktail as previously explained [44]. Isolation of chromatin-bound proteins was performed using cytoskeletal buffer (10?mM PIPES-KOH (pH 6.8), 100?mM NaCl, 300?mM sucrose, 3?mM MgCl2, 0.5?mM PMSF, 0.1?mM glycerolphosphate, 50?mM NaF, 1?mM Na3VO4, containing 0.1% Nonidet P-40 and protease inhibitor cocktail) as previously explained [45]. Chromatin immunoprecipitation (ChIP) and biotin-labeled DNA pull-down assay ChIP assays were performed as previously explained [43]. DNA was purified from immunoprecipitates by STAT1 antibodies by phenol/chloroform extraction and utilized for PCR. The primers specific for the and promoters were: promoter PI-103 (?201 to ?89) probes, putative STAT1-binding site-mutant probes, and IL-2 promoter (?100 to ?69) probes were synthesized by Sangon. The mutated probes for the PI-103 putative STAT1-binding sites shown in the lower case were ?191 TTTTCCTGGGG ?181 to ?191 ggggaagtttt ?181 and ?110 TTCTGGAAAA-101 to ?110 ggagttcccc ?101. Nuclear extracts were incubated with biotinylated promoter DNA at 4?C overnight. The DNA-protein complexes were precipitated by streptavidin-conjugated agarose beads (Yeasen), washed with lysis buffer five occasions and boiled in 1??Laemmli sample buffer before being subjected to immunoblotting analysis. In situ Proximity Ligation Assay (PLA) and Immunofluorescence Microscopy The conversation between ADAP and STAT1 was detected in formalin-fixed, paraffin-embedded (FFPE) spleen sections by PLA using a Duolink in situ detection kit (Sigma) according to the PI-103 manufacturers instructions. For immunofluorescence microscopy, FFPE tissue slides (5 m) were deparaffinized.