Hrp labeled goat anti mouse igg

DC 2.4 cells were seeded in 24-well plates at 500,000 cells/well and then transfected with 1 μg of mRNA to form cell monolayers using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). After 6 h, the medium was replaced with DMEM supplemented with 3% FBS. Following 24 h post-transfection, cells were fixed with ice-cold acetone for 10 min and incubated with a polyclonal mouse anti-CHIKV antibody (prepared in our laboratory) and then FITC-labeled goat anti-mouse IgG (Abcam). Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Fluorescence was observed using a fluorescent microscope (Olympus).
After 16–26 h post-transfection, cells were lysed with RIPA buffer plus proteinase inhibitor (Sigma), clarified by centrifugation at 12,000 × g, and then 5 × SDS loading buffer was added, and cells were kept at 95°C for 10 min. The lysates were run on a 10% NuPAGE Bis-Tris gels (Invitrogen) followed by transferring proteins to a nitrocellulose membrane. The membrane was incubated with a polyclonal mouse anti-CHIKV antibody (prepared in our laboratory) and HRP-labeled goat anti-mouse IgG (Abcam). DC 2.4 cells with lysing buffer and E2 protein were used as negative and positive controls (ProSpec), respectively. Blots were developed using ECL reagents (GE).

Nintedanib (>99%) was purchased from HWRK Chem Co., Ltd. (Beijing, China). For each experiment, Nintedanib was freshly prepared by dissolving it in DMSO (Sigma-Aldrich, USA). Bleomycin (BLM) was acquired from Nippon Kayaku (Tokyo, Japan). Wnt3a was purchased from Peprotech (Texas, USA). TRIzol reagent and DEPC-treated water were obtained from Thermo Fisher Scientific corporation (Waltham, USA). RNase, DNase, and DNA Away H₂O were purchased from Beyotime Biotechnology (Beijing, China). FastKing gDNA Dispelling RT SuperMix was obtained from Tiangen Biotech Co., Ltd. (Beijing, China). The inhibitor KX2-391, RIPA lysis buffer (middle) and the BCA kit were purchased from Beyotime Biotechnology (Beijing, China). The primary antibodies described in the study included anti-fibronectin, anti-collagen I, and anti-β-tubulin antibodies (Affinity Biosciences, USA); anti-GAPDH, anti-p(Y416)-Src, and anti-Src antibodies (Cell Signalling Technology, USA); anti-α-actin antibody (Santa Cruz Biotechnology, USA); anti-β-catenin and anti-lamin B1 antibodies (ProteinTech Group, China); and anti-p(Y654)-β-catenin antibody (Immunoway Biotechnology, China). The secondary antibodies HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were from Abcam (Cambridge, UK).

Samples collected from Sf9 cells infected with rBVs were treated with 5×SDS Loading Buffer for 10 min at 100 °C and separated using 12% SDS-PAGE gels. For Western blot analysis, samples separated through SDS-PAGE were transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilin-p, Millipore, USA), after which, the membranes were blocked for 1 hour in 5% milk in Tris-buffered saline with Tween 20 (TBST). Two different antibodies were used to detect the presence of M, F, H and N: mouse polyclonal antibody for M protein and sheep polyclonal antibody for N, F, and H protein. HRP-labeled goat anti-mouse IgG (1:10000 dilution; Abcam, USA) and HRP-labeled rabbit anti-sheep IgG (1:10000 dilution; Bioworld) were used as secondary antibodies. The membranes were washed in TBST and visualized using Western Blue stabilized substrate (Promega, USA) to detect alkaline phosphatase.