Platelet agglutination rate maximum [PAG (M)] and PAG at 1 min [PAG (1)] were examined and an agglutination assay was conducted using a platelet aggregation apparatus (Order no. wild-type. Platelet agglutination was also significantly inhibited in the LRRFIP1?/?mouse model compared with the wild-type. LRRFIP1 knockout significantly decreased the IIb3 levels in platelets undergoing convulxin treatment. In conclusion, LRRFIP1 treatment brought on platelet agglutination and LRRFIP1 gene knockout inhibited platelet agglutination. In addition, LRRFIP1 gene knockout significantly decreased the levels of IIb3. This suggests that LRRFIP1 my be applied to patients in a clinical setting to trigger platelet agglutination Chlorpheniramine maleate in inflammatory diseases and atherothrombotic diseases. strain (cat. Chlorpheniramine maleate no. GSB013; ZonHon Biopharma Institute Inc., Changzhou, China) was cultured in lysogeny broth (LB) liquid medium (cat. no. L3152; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Full-length recombinant LRRFIP1 protein was purified from the total crude extract of BL21 as previously explained (20). Briefly, the BL21 strain was cultured in the LB culture Chlorpheniramine maleate medium and then re-suspended in lysis liquid (cat. no. T9424; Sigma-Aldrich; Merck KGaA) to obtain total protein, which was purified using glutathione-S-transferase soluble protein (Sangon Biotech Co., Ltd., Chlorpheniramine maleate Shanghai, China). The prokaryotic-expressed proteins were extracted using glutathione sepharose 4B beads (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s protocol. The highly purified LRRFIP1 was identified as high-density bands around the SDS-PAGE images. Briefly, the concentration of the obtained recombinant LRRFIP1 was decided using a bicinchoninic acid protein assay kit according to the manufacturer’s protocol. A total of 0.2 g protein lysates were separated with SDS-PAGE on a 15% gel. Animals A total of 20 BALB/C mice with 6C8 week-old (10 male and 10 female), weighting from 25 to 35 g were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All mice used in the present study were housed in cages (5 mice/cage) under the same conditions, including a controlled environment at 22C with 50% humidity and a 12 h light/dark cycle. The food, water and bed linens were sterilized, and the mice experienced free access to food and water. All animal experiments were approved by the Ethics Committee of Daping Hospital (Chongqing, China) and all mice were handled in accordance with the Guidelines for Care and Use of Laboratory Animalsby the National Institute of Health (21). Establishment of plasmids and a mouse model of LRRFIP1 gene knockout To produce LRRFIP1 expression plasmids the targets of the LRRFIP1 gene were designed as detailed in Fig. 1A. Embryonic stem cells (cat. no. CRL-11379; American Type Culture Rabbit Polyclonal to UBA5 Collection, Manassas, VA, USA) were used to clone the LRRFIP1 gene according to the previously published studies Chlorpheniramine maleate (16,17). Several strains of the LRRFIP1 gene are outlined in Fig. 1B, andexon 2 was the most conservative. Therefore, the LRRFIP1 gene knockout mice were established by synthesizing the mutated exon 2 gene. Among the outlined LRRFIP1 genes, three target gene sequences were selected and cloned into the px458 plasmid (cat. no. 3683466; BioVector NTCC Inc., Beijing, China) to construct LRRFIP1?/? expression plasmids by employing the BamH I and EcoR I restriction enzymes (Fig. 1C); these plasmids were used to develop the LRRFIP1 knockout mouse model. Target gene 3 illustrated the highest-density band (Fig. 1D), which was selected to establish the LRRFIP1?/? expression plasmid according to previous studies (16,17). Open in a separate window Physique 1. Processes of gene selection and plasmid construction and the establishment for any mouse model of LRRFIP1 gene knockout. (A) The targeting gene for LRRFIP1. (B) Several strains of the targeting.