Horseradish peroxidase hrp conjugated anti rabbit igg

Experimental and transfected cells were collected, homogenized, and lysed using TPEB^® assay reagent (Bio Sharp, Wuhan, China), containing 0.5 mM of phenylmethanesulfonyl. Protein concentration was determined by BCA protein assay (Takara, Shanghai, China). Samples of 25 μg protein were fractionated by SDS-PAGE in 10% gradient Tris-glycine precast gels (Solarbio, Shanghai, China) and transferred to methanol activated-polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 h in a blocking solution of QuickBlock^TM Western (Beyotimes Biotech, Wuhan, China) for 15–20 min at room temperature with gentle shaking at 4 °C with overnight incubation in primary antibodies against p65 (1:1000; Proteintech, Wuhan, China), p-p65, IkBα, p-IkBα, and β-actin (1:500, 1:800, 1:500, 1:600 and 1:1000 dilution rates respectively; Abcam, London, UK), TLR4, (1:500, Proteintech, China), IRAK4, MyD88 and TRAF6 (1:400, 1:300:1:500, Beyotimes Biotech, Shanghai, China). Subsequently, the labelled proteins were visualized by incubation with a horseradish-peroxidase (HRP) conjugated anti-rabbit IgG (1:50,000; Abcam, London, UK) followed by development with a chemiluminescence substrate (ECL) for HRP (Thermo Fisher Scientific, San Diego, CA, USA). The images of western blots were captured by GE ImageQuant.2.11.

Immunoplates (Cat. No. 439454, ThermoFisher Scientific) were coated overnight at 4°C with various amounts (1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, and 0 ng/mL) of MTB protein (MPT64, Cat. No. AB225589; Abcam) in 100 μL of 0.05 M carbonate–bicarbonate buffer (Cat. No. C3041-100CAP; Sigma-Aldrich, MO, United States) per well. After extensive washing with PBS containing Tween 20 (PBST), the plates were blocked for 1 h with 5% bovine serum albumin (BSA) in PBS with 0.1% Tween-20Subsequently, a 1:1,000 diluted MTP64 Ab (Cat. No. AB193435; Abcam) in 5% BSA in PBST was added to each well and incubated for 1 h. Following another round of extensive washing with PBST, the bound antibodies were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5,000; Cat. No. #7074; Cell Signaling Technology, MA, United States) detection antibody for 1 h. After thorough washing with a wash buffer, stabilized chromogen (TMB solution, Cat. No. 34028; ThermoFisher Scientific) was added. The immunoplates were allowed to react for 10 min, and the reaction was stopped by adding 2 N H₂SO₄. The optical density (OD) value was measured at 450 and 650 nm using a microplate reader (SpectraMax, Molecular Devices, San Jose, CA, United States).

Eagle's Minimum Essential Medium (MEM), Nutrient Mixture Ham's F12 medium (F12), fetal bovine serum (FBS), and other supplements for cell culture were purchased from Gibco (Gaithersburg, MD, USA). Retinoic acid (RA), 1-methyl-4-phenylpyridinium (MPP⁺), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primers for real-time RT-PCR were synthesized by and purchased from Biolegio (Nijmegen, Netherlands). Antibodies against Akt, phosphorylated Akt, phosphorylated mTORC1, and tyrosine hydroxylase (TH) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against eEF1A2 and anti-β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were purchased from Abcam (Cambridge, UK) and Invitrogen (Eugene, OR, USA), respectively.