Horseradish peroxidase conjugated secondary antibody

BEAS-2B cells were treated with Baicalein (2.5 μM) or vehicle (DMSO) for 30 min, followed by TNF-α (10 ng/mL) exposure for 60 min and 2 h to collect total protein and nuclear and cytosolic proteins, respectively. Lung (100 μg) and cellular (50 μg) protein samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories Inc, USA). After blocking in blocking buffer (5% milk in Tris-buffered saline containing 0.05% Tween 20 [TBST]) for 1.5 h at room temperature, the membranes were incubated with different primary antibodies overnight at 4 °C. Afterwards, the membranes were washed in TBST and reacted with secondary horseradish peroxidase-conjugated antibody (Santa Cruz, CA, USA; 1:3000) for 1–2 h at room temperature. Blots were then visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories Inc, USA). The densities of the immunoreactive bands were analyzed using ImageJ software (NIH, Bethesda, MD, USA). Antibodies against IκBα (1:300), NF-κB p65 subunit (1:300), and lamin B (1:300) were purchased from Santa Cruz Technology (Santa Cruz, CA, USA). Antibodies against P-IκBα (1:1,000) and inducible nitric oxide synthase (iNOS, 1:1,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

The expression of BDNF protein in the hippocampus was evaluated by Western blot analysis. The concentrations of proteins extracted with TRizol reagent (Thermo Fisher Scientific, Inc., Tokyo, Japan) were determined using Bradford’s method with bovine serum albumin (BSA) as a standard. Each amount of proteins was subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes (0.45 µm, Merck Millipore, Billerica, MA, USA). The membranes were blocked with 3% BSA in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) for 1 h at room temperature, and incubated overnight at 4 °C with primary antibodies against BDNF (1:3000; Abcam, Cambridge, MA, USA) and tubulin (1:5000; Abcam). Next, the membrane was washed with TBST three times and incubated with secondary horseradish peroxidase-conjugated antibody (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. The protein bands were visualized using the EzWestLumi plus kit (ATTO, Tokyo, Japan) and AE-9300 Ez-Capture (ATTO). The intensity of protein bands was analyzed using ImageJ software (NIH Image, Bethesda, MD, USA).

The cells were plated in 6-well plates at a density of 1 × 10⁶ cells per well. After incubating overnight, the cells were treated with HPA3P at the indicated concentration. After incubating for 6 h or 24 h, the cells were scraped, and the proteins were quantified by the Bradford method. The proteins were resolved on SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, USA). The blots were blocked with TBST containing 5% skim milk for 1 h, after which the membranes were incubated overnight at 4°C with primary antibodies against cleaved caspase-3 (#9664), PARP (#9542, Cell Signaling Technology, MA, USA), HMGB1 (ab18256, Abcam, Cambridge, UK), and GAPDH (SC-25778, Santa Cruz, CA, USA). The blots were then washed with TBST buffer and incubated with a secondary horseradish peroxidase-conjugated antibody (Santa Cruz, CA, USA). The reaction was detected using a Clarity™ Western ECL Substrate (BioRad, CA, USA).

Cells were harvested in RIPA buffer (Thermo Scientific) with protease and phosphatase inhibitors (Roche Diagnostics). Immunoblotting was done after sodium dodecyl sulfate–polyacrylamide gel electrophoresis on gradient 4–15% gels (Biorad) at 30 μg protein and transferred to polyvinylidene fluoride membranes. All antibodies besides the secondary horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology) were purchased from Cell Signaling Technologies: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), p44/42 MAPK (ERK1/2), phospho-p38 (Thr180/Tyr182), p38, phospho- MEK (Ser 217/221), MEK, RhoC, RhoA, ß-actin. SuperSignal West Pico Luminol/Enhancer Solution was purchased from Thermo Scientific.

Cell protein samples (50 μg) were subjected to 10% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories). After being blocked in blocking buffer (5% milk in tris-buffered saline containing 0.05% Tween 20) for 1.5 h at room temperature, membranes were incubated with different primary antibodies overnight at 4 °C. The membranes then were washed in TBS-T and reacted with secondary horseradish peroxidase-conjugated antibody (Santa Cruz, CA, USA;1:5000) for 1–2 h at room temperature. Blots were then visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories). The density of the immunoreactive bands was analysed using Image J software (NIH, Bethesda, MD, USA).