Westernbright ecl enhanced chemiluminescent substrate kit

EfSEV and VF protein concentrations were measured using a BCA kit (Solarbio, Beijing, China). An equivalent volume (10 µg) of EfSEV, VF, NC and NCs was separately mixed with 4× Loading Buffer (Solarbio, Beijing, China) and vortexed. The mixture was heated in a water bath at 100 °C for 10 min. The protein components were resolved in a 12% SDS-PAGE gel, and the protein bands were transferred to a polyvinylidene difluoride membrane (Merck-Millipore, Germany). After 1 h of blocking with 0.01% Tween-20 in PBS (PBST) containing 5% skimmed milk, the membrane was incubated with anti-heat shock proteins 70 kDa (Hsp70; Proteintech Group Inc., Rosemont, IL, USA) overnight at 4 °C. Thereafter, the membrane was washed in PBST and incubated with horseradish protein-conjugated antibodies (Proteintech Group) for 1 h at room temperature. The membrane was treated with the WesternBright™ ECL enhanced chemiluminescent substrate kit (Advansta Inc., San Jose, CA, USA) according to the manufacturer’s instruction and visualized using the Luminescent Image Analyzer Amersham Imager 600 series (GE Co., Boston, MA, USA).

BV2 cells were seeded in a 6-well plate (n = 3), the supernatant was removed after 24 h LPS stimulation. RIPA buffer supplemented with protease inhibitor cocktail was added to the BV2 cell lysate and homogenized. Twenty μg of total protein was fractionated by SDS-PAGE (12% resolving and 6% stacking) and immunoblotted with phospho-p38 (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p44/42 (Erk1/2) (Thr202/Tyr204), NF-κB p65, MyD88, [PVDF membrane; diluted 1:1000 in 5% w/v BSA, 1X TBS, 0.1% Tween-20], iNOS [NC membrane; dilution 1:700 in 5% w/v BSA, 1X TBS, 0.1% Tween-20], β-actin [dilution 1:3000 in 5% w/v BSA, 1X TBS, 0.1% Tween-20], and anti-rabbit IgG, HRP-linked antibody [1:3000 dilution in 5% w/v skim milk, 1X TBS, 0.1% Tween-20]. Immunoblotted protein bands were visualized with enhanced chemiluminescence with WesternBright™ ECL enhanced chemiluminescent substrate kit (Advansta Corporation, San Jose, CA, USA) following manufacturer’s instructions. Quantitative determination of protein expression was performed using Image Lab™ software v5.2.1 (Gel Doc™ XR+, Bio-Rad, CA, USA).