Ab62566

AMΦ and lung tissues were lysated in radio-immunoprecipitation lysis buffer (RIPA), protease inhibitors (Roche, Mannheim, Germany) and phenylmethylsulfonyl fluoride (PMSF). Protein concentrations were subsequently determined by standard BCA assay. After addition of 6 × sodium dodecyl sulfate (SDS) loading buffer, equivalent amounts of protein were heated (100 °C; 5 min) and separated by gel electrophoresis using a 10% SDS-polyacrylamide electrophoresis gel. Resolved proteins were then transferred to a nitrocellulose membrane and blocked with Tris-buffered saline containing Tween-20 (TBST) and 5% nonfat milk (1 h; 24 °C). Nitrocellulose membranes were incubated overnight at 4 °C with primary antibody against TLR3 (ab62566; Abcam, Hong Kong, China), NF-κB p65 (ab7970, Abcam, Hong Kong, China), Abcam, MIP-2 (ab25130, Hong Kong, China), PCNA (ab18197, Abcam, Hong Kong, China) and β-actin (ab8226, Abcam, Hong Kong, China). The membranes were washed in TBST three times, incubated with secondary antibody (926-32221 IRDye 680 mouse-anti-rabbit secondary antibody; Licor Biosciences, Lincoln, NE, USA) for 1 h at 37 °C and then washed in TBST three additional times. The membranes were determined by using an Odyssey image analysis system (Licor Biosciences). Western blots were quantitated using Quantity One software (Bio-Rad, Foster City, CA, USA) and normalized to β-actin and PCNA signal.

Heart tissue and cultured cardiomyocytes were lysed in RIPA buffer containing protease inhibitors at 4°C. Lysates were collected and determined with protein concentration using a bicinchoninic acid kit. For Western blot, 20 µg of total proteins was separated on the SDS‐PAGE gel and transferred onto PVDF membrane (Immobilon‐P Transfer Membrane, Millipore Corp). The membrane was then blocked with PBST buffer containing 5% non‐fat dried milk for 1 hour and further incubated overnight at 4°C with primary antibodies against TLR3 (ab62566, Abcam), TLR4 (NB100‐56566, NOVUS) or FOXC1 (#8758, Cell Signaling Technology). After that, the membrane was incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies (sc‐2004, sc‐2005, Santa Cruz Biotechnology) for 1.5 hours at room temperature and visualized by chemiluminescence reagents. GAPDH (ab8245, Abcam) was used as a loading control.

Cells grown on coverslips were fixed with acetone/methanol (1:1) for 20 min on ice and washed in 3 changes of PBS (5 min each). Blocking was performed at room temperature for 30 min, in 3 changes of 1% BSA/0.1% Tween 20/PBS. An anti-TLR3 antibody (ab62566, Abcam; Cambridge, UK), diluted 1:200 in blocking buffer, was applied overnight at 4°C. After 3 washes in blocking buffer, a donkey anti-rabbit IgG-labeled secondary antibody was added in blocking buffer for 1 h at room temperature. Cells were washed in blocking buffer, mounted in antifade medium, and visualized and photographed using fluorescence microscopy.

Cells were lysed using RIPA Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) with addition of protease and phosphatase inhibitor (#5872, Cell signaling). Following lysis, cells were incubated on ice for 10 min and centrifuged for 10 min at 4° at 16,000 × g. In all, 40 μg of each protein lysate was electrophoresed using SDS-PAGE, transferred to nitrocellulose membrane, blocked with 5% non-fat milk, and incubated overnight with following antibodies; anti-BCL9 (1:1000 dilution, ab37305, Abcam), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), TLR3 (1:500 dilution, ab62566, Abcam), β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories), CD44(1:1000 dilution, #3570, Cell signaling), Flag tag (1:1000 dilution, A8592, sigma), Axin2 (1:1000 dilution, #2151, Cell signaling), p65 (1:1000 dilution, #8242, cell signaling), LaminB1 (1:1000 dilution, sc-6216, Santa Cruz), and GAPDH (1:1000 dilution, ab9485, Abcam). Secondary antibodies conjugated to horseradish peroxidase were purchased from Santa Cruz Biotechnology (1:5000 dilution, sc2020, Santa Cruz) and Cell signaling (1:5000 dilution, #7074, #7076, Cell signaling).