Celllytic mt mammalian tissue lysis reagent

CellLytic MT Mammalian Tissue Lysis Reagent (C3228, Sigma-Aldrich), mixed with a protease inhibitor cocktail (1:100, EMD Millipore), was added to frozen tissue in 2-mL Lysing Matrix D tubes (P000912-LYSK0, Precellys) to be homogenized using a MINILYS personal homogenizer (Bretin Instruments). Homogenized tissue lysates were centrifuged and supernatants were used for protein concentration determinations with a Pierce TM BCA protein assay (23225, Thermo Fisher Scientific).

The Western blot method was performed as previously described (Lu et al., 2018 (link)). The hippocampal tissues were homogenized on ice in CellLytic MT mammalian tissue lysis reagent (C3248, Sigma-Aldrich, St. Louis, MO, United States) containing protease and phosphatase inhibitor cocktails (P3850, Sigma-Aldrich, St. Louis, MO, United States). The homogenate was centrifuged at 10,000 ×g for 15 min at 4°C and the protein concentration was further determined by using an enhanced BCA protein assay kit (Thermo scientific, Waltham, MA, United States). Protein samples (30 μg/sample) were electrophoresed on a 10% Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS–PAGE) and transferred onto PVDF membranes (Millipore, Burlington, MA, United States). The membranes were blocked for 1 h using 5% non-fat dry milk in Tris-Buffered Saline (TBS) containing 0.5% Tween-20 (TBST), then probed with specific primary antibodies against phosphor ERK1/2, ERK 1/2, phosphor CREB, and CREB, BDNF (1: 500), and β-actin (Cell Signaling Technology, Danvers, MA, United States) overnight at 4°C. After thoroughly washed with PBST (PBS with 0.1% Tween 20), the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for another 1 h. The protein bands were visualized with ECL prime kit (GE Healthcare, NA, United Kingdom).

Mice were stimulated by intraperitoneal injection with 100μg of polyinosinic:polycytidylic acid (P1530, Sigma, St. Louis, MO). After 24 hours stimulation, spleens were isolated and homogenized by sonication and tissue homogenates were lysed with CellLytic MT Mammalian Tissue Lysis Reagent (Sigma, St. Louis, MO). Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels, transferred to polyvinylidene fluoride membranes, blocked with Odyssey Blocking Buffer (LI-COR, Lincoln, NE) probed with monoclonal mouse anti-TLR3 (Novus Biologicals, Littleton CO) and polyclonal goat anti-tubulin Santa Cruz Biotech, Santa Cruz, CA) primary antibodies and IRDye 800CW and IRDye 680 RD secondary antibodies (LI-COR, Lincoln, NE). Membranes were scanned with the LI-COR Odyssey Scanner (LI-COR, Lincoln, NE). Protein was quantified using LI-COR Odyssey 3.0 Software.