Horseradish peroxidase conjugated anti rabbit igg

The mice sacrificed by anesthesia using CO2. Total protein was extracted from mouse liver using protein lysate (RIPA: PMSF = 100:1). The total protein was determined by the BCA protein assay kit (Thermo). Proteins in samples were fully denatured by heating in 4× loading buffer at 100 °C for 10 min, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA). Membranes were blocked with non-fat milk and probed with primary antibodies (anti-TfR1, anti-FPN1, and anti-GAPDH) (Santa Cruz). Membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz). After washes three times the blots were visualized by enhanced chemiluminescence with chemiluminescence system machine (Clinx Company, ChemiScope 6300). Gray value of Western blot strips was quantified with Image J software (National Institutes of Health).

MGC80-3 and AGS cells were harvested at the 5^th day post infection. Then cells were lysed in 2× SDS Sample Buffer (100 mM Tris-HCl, pH = 6.8, 10 mM EDTA, 4% SDS, 10% Glycine). The concentration of protein in the cell lysate was determined using the BCA protein assay kit (Pierce Biotechnology, Cat no. 23235). Totally, 30 μg of protein was loaded on per lane, followed by separation using SDS-PAGE, and transfer to PVDF membrane. Then the membrane was subsequently incubated with primary antibodies, including rabbit anti-human-GGCT (Sigma, Cat no. HPA020735, 1:500), rabbit anti-human-GAPDH (Proteintech Group, Inc., Cat no. 10494-1-AP, 1:60000), overnight at 4 °C, which was followed by incubation with secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz, Cat no.SC-2054, 1:5000), for 1 h at room temperature. Blots were visualized using ECL Test Kit (Amersiam, Cat no. RPN2132). GAPDH was used as the internal control. Density analysis was carried out using Quantity One software (BioRad).

The spinal cord was lysed in ice-cold RIPA buffer with a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO). Tissues were homogenized using a tissue homogenizer. Spinal cord samples were loaded into the 10% polyacrylamide gels and then were electrotransferred onto a nitrocellulose membrane. The membranes were blocked with 10% non-fat dry milk in PBS with 0.3% Tween for 1 hour and then incubated overnight at 4°C with rabbit anti-TRPV1 antibody (1:500; Alomone Labs, Jerusalem, Israel), or GAPDH antibody (1:5000; Sigma Aldrich, St. Louis, MO) diluted in PBS. The membrane was then incubated in horseradish peroxidase-conjugated anti-rabbit IgG (1:5000; Santa Cruz Biotechnology, Dallas, TX) for 2 hours at room temperature. The antigen–antibody complexes were visualized using an enhanced chemiluminescence detection reagent (Bio-Rad Laboratories, Hercules, CA). Bands were scanned using a densitometer (GS-700; Bio-Rad Laboratories, Hercules, CA), and quantification was performed using NIH Image J software.

The cells were harvested, washed twice with cold phosphate-buffered saline (PBS) and lysed in buffer [50 M Tris-HCl (pH 7.4), 150 M NaCl, 2 M EDTA and 1% NP-40], containing protease inhibitors. The protein concentration was quantified using a bicinchoninic acid protein measurement kit (Shenneng Bocai Biology, Co., Ltd., Shanghai, China). A total of 30 μg extract was subjected to SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane, which was blocked with 5% (w/v) dried skimmed milk-tris-buffered saline and tween 20 [TBST; 10 M Tris-HCl (pH 8.0), 150 M NaCl and 0.05% Tween 20] for 1 h at room temperature. Subsequently, the membrane was incubated for 2 h with antibodies against RECK (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA; at 1:1000 dilution) and MMP-2 (Santa Cruz Biotechnologies, Inc.; at 1:1000 dilution), respectively, in TBST with 5% skimmed milk. Subsequent to being washed in TBST, horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Inc.) was used as a secondary antibody (1:10,000, in TBST with 2% bovine serum albumin incubated for 1 h). Each sample was also probed with an anti-GAPDH antibody (Santa Cruz Biotechnology, Inc.) as a loading control. Protein bands were visualized by the Alpha Imager 2200 system (Alpha Innotech, San Leandro, CA, USA).

Immunoblot analysis of CMV CP extracted from virus-infected plants was performed according to the method described previously (Du et al., 2014a (link)). Briefly, upper systemically-infected leaves were harvested at 14 dpi and used for extraction of total soluble proteins, which were separated in a 15% SDS–PAGE gel, and transferred onto a nitrocellulose membranes. Membranes were incubated with the polyclonal anti-CP serum (Agadia). For analyzing GFP protein, total soluble protein was extracted from leaves expressing GFP or GFP-2b fusion proteins at 5 dpi. Primary antibody binding on membranes was detected using horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz) and ECL substrate (Thermo-Fisher).