Sqab1501

Phorbol-12-myristate-13-acetate (PMA), gelatin, Triton X-100, Coomassie brilliant blue R-250 was purchased from Sigma-Aldrich, MMP-9 inhibitor (SB-3CT) and NF-κB inhibitor (sc-75741) were purchased from Selleck. Recombinant human MMP-9 protein and Recombinant mice MMP-9 protein were purchased from R&D systems. Recombinant human pro-MMP-9 protein were purchased from ProSpec. Commercialized DENV2-NS1 protein were purchased from Native Antigen. Trizol reagent was purchased from Ambion. Lipofectamine 2000 reagent was purchased from Invitrogen. Human MMP-9 ELISA kit was purchased from BD Biosciences. Membrane maker Dil (D8700) was purchased from Solarbio. NF-κB Pathway Sampler Kit (#9936T), Tight Junction Antibody Sampler Kit (#8683T), Cadherin-Catenin Antibody Sampler Kit (#9961T), and Antibodies against TIMP-1 (8946S) were purchased from Cell Signaling Technology. Antibody against DENV-NS3 (GTX124252) were purchased from Genetex. Antibodies against DENV2-NS1 were purchased from Arigo biolaboratories (SQab1501). Antibodies against Flag (F3165) and HA (H6908) were purchased from Sigma. Anti-β-actin antibody (66009) were purchased from Proteintech. Rabbit IgG and Mouse IgG were purchased from Invitrogen. Anti-Mouse IgG Dylight 649, Anti-Mouse IgG Dylight 488, Anti-Rabbit IgG Dylight 649, and Anti-Rabbit IgG FITC were purchased from Abbkine.

Cells were lysed using lysis buffer containing 1% (v/v) protease inhibitor cocktail, 1 mM Phenylmethylsulfonyl fluoride, and 1 mM DTT. Total proteins were extracted by centrifugation at 12000 × g at 4°C for 15 min. Input proteins were quantified using Coomassie brilliant blue (Sigma-Aldrich, MO, USA) staining and denatured using 5 × loading buffer and boiled for 10 min. For electrophoresis, equal amount of the proteins was loaded and subjected to SDS-PAGE, after which the proteins were electrically transferred to a PVDF film (Amersham, Ayesbury, UK). The membranes were then blocked in TBST (pH 7.4, 0.5% Tween20) with 5% skim milk powder for 2 hours, and then incubated with the primary antibodies in TBST-5% BSA at 4°C overnight. Then, the membranes were washed with TBST and incubated with secondary antibodies at RT for 1 h, after which the membrane were visualized with an ECL kit (KeyGEN, Nanjing, China). Antibodies were purchased from the indicated companies and used at dilution 1:1000, including primary antibody: rabbit anti-NLRP12 (ab105409, Abcam), rabbit anti-β-actin (13E5, CST), mouse anti-Dengue virus NS1 (SQab1501, Arigo), mouse anti-Dengue virus NS5 (SQab19173, Arigo), rabbit anti-PRDM1 (C14A4, CST), rabbit anti-Flag (D6W5B, CST). Secondary antibody: anti-rabbit IgG mAb (L27A9, CST), anti-mouse IgG mAb (D3V2A, CST).