Hrp labeled goat anti rabbit igg h l

Silibinin, curcumin, methylthiazolyldiphenyl-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). Anti-leptin receptor antibody and bicinchoninic acid protein assay kits were purchased from Abcam (Cambridge, UK). Monoclonal antibody against Beta-actin and HRP-labeled goat anti-rabbit IgG (H+L) were purchased from Cell Signaling Technology (Danvers, MA, USA). The polyvinylidene difluoride membrane was purchased from PerkinElmer (Waltham, MA, USA). An enhanced chemiluminescence kit (ECL) was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

When observing the different concentrations of TP influence on TLR4, cell
suspension in the logarithmic phase was seeded in a 6-well plate (2 mL, 5 ×
10⁵ cells per mL), and different concentrations of TP (5, 10, 20,
and 40 μg/mL) were incubated for 24 h. TP (40 μg/mL) was chose to treat cells
for 0, 6, 12, and 24 h. Cells were also lysed with ice-cold lysis buffer (50-mM
Tris-HCl, pH 6.8, 100-mM 2-mercaptoethanol, 2% w/v sodium dodecyl sulfate, and
10% glycerol). Proteins were separated with 10% sodium dodecyl
sulfate–polyacrylamide electrophoresis and then transferred onto a
polyvinylidene difluoride (PVDF) membrane at 100 V for 1.5 h. Proteins were
treated with Tris-buffered saline and Tween which contained 5% non-fat dried
milk for 1 h. Blots were incubated with primary antibodies (Rabbit Anti-human
TLR4, 1:800, Cell Signaling) at 4°C overnight. This was followed by incubation
with secondary antibodies (HRP-labeled Goat Anti-Rabbit IgG (H + L), 1:2000,
Cell Signaling) at room temperature for 2 h. After being washed with PBST
(phosphate buffered saline + 1% Tween 20), blots were analyzed using Gel-Doc 200
(Bio-Rad). Bio-Rad (Hercules, CA, USA) GAPDH. GAPDH: glyceraldehyde phosphate
dehydrogenase was used as the internal reference.