β actin primary antibody

The liver tissues were homogenized in a cold RIPA lysis buffer to prepare hepatic homogenate. Equal amounts of protein determined using BCA kits were separated on 10% SDS-PAGE; transferred onto PVDF membranes; blocked with 5% skim milk; and incubated with NLRP3, TLR4, NF-κB, JNK or β-actin primary antibodies (Cell Signaling, Danvers, MA, USA) overnight at 4 °C. Afterwards, the membrane was washed with TBST and incubated with a secondary antibody (Cell Signaling). β-actin was considered the internal control. The bands of protein were observed and quantified using a chemiluminescence (ECL) localization reagent and a Tanon GIS analysis [24 (link)].

Pomegranate fruits were obtained from Lintong, Shaanxi province of China, and the extraction and purification of PPPs were performed in our laboratory (20 (link), 21 (link)). Detailed methods and results have been presented in the previous article (28 (link)). The polyphenol content of PPPs was 57.09%. The main polyphenol compounds were gallic acid, Punicalagin (Punicalagin-α and Punicalagin-β), catechin, chlorogenic acid, epicatechin, and ellagic acid. The yielding of PC, which is a major component of PPPs, was 464.48 mg/g, and the yielding of EA was 71.50 mg/g. The concentration of the other components of PPPs – catechin, gallic acid, epicatechin, and chlorogenic acid – was 45.14, 38.24, 35.28, and 8.85 mg/g, respectively.
Punicalagin, ellagic acid, and LPS (Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture reagents and fetal bovine serum were purchased from Gibco BRL (Rockville, MD, USA). TAK-242 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Bay11-7082, MG-132, ROS, and NO assay kits were purchased from Beyotime Biotechnology (Beijing, China). TLR4, P65, p-IκBα, IκBα, histone 1, and β-actin primary antibodies for Western blot analysis were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

The primary antibody for ERβ was purchased from abcam (Cambridge, MA, USA). The β-actin primary antibody was produced by Cell Signaling (Beverly, MA, USA). Herceptin is manufactured by Genentech Inc. (San Francisco, USA). ON-TARGETplus siRNA to ESR2, ON-TARGETplus SMARTpool siRNA to ERBB2 (a pool of four siRNA), and a negative control siRNA were purchased from GE Healthcare Dharmacon Inc. (Pittsburgh, PA, USA). All siRNAs were transiently transfected into SKBR3 cells using FuGENE HD transfection reagent (Roche, Switzerland) per manufacturer's instructions. The pAcGFP1-C1-ER beta plasmid, a GFP-tagged ESR2 overexpression plasmid, was a kind gift from Dr. Ratna Vadlamudi (UTHSCSA). This plasmid was transfected into SKBR3 cells using FuGENE HD, and G418 (Life Technologies) was used to stably select transfected cells. Clones were selected and the two with the highest ESR2 expression (SKBR3-ERB#3 and SKBR3-ERB#5), as determined by RT-PCR, maintained in IMEM plus 10% FBS and 500 ug/ml G418. Dr. Carla Van Den Berg (University of Texas at Austin) generously provided the pGFP vector, which was also stably transfected into SKBR3 cells (SKBR3-GFP) using FuGENE HD.

Cellular protein was analyzed by Western blotting as reported [21] (link). Briefly, cellular protein was extracted with detergent, and the protein concentration of the lysates was determined for each sample with the BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein were separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Pierce Biotechnology, Inc. Rockford, IL, USA). After blocking with 5% skim milk, the PVDF membranes were incubated with p53, p21, cyclin E1, Fas-L, cleaved caspase 3,8,9 or β-actin primary antibody (all rabbit IgGs; 1∶1000; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. The membranes were washed three times in PBST (10 mM NaH₂PO₄, 130 mM NaCl, 0.05% Tween 20) and then probed with a horseradish peroxidase-conjugated secondary antibody (goat anti rabbit IgG; 1∶5000; Molecular Probes, Invitrogen) for 1 h. Protein expression was visualized by the enhanced chemiluminescence (ECL) western blotting detection reagent (GE Health Care Bio-Sciences, Piscataway, NJ, USA).