Anti p iκbα

Lymphocytes inside the blood from rabbits were isolated through lymphocytes separation medium (Mediatech, Manassas, USA). The isolated lymphocytes were sonicated and centrifuged. Proteins in the supernatants of cell lysates were separated by 12% polyacrylamide gel electrophoresis. After SDS-PAGE, the isolated proteins were transferred onto polyvinylidene fluoride membranes (Sigma). The membranes were then washed twice with cold Tris-buffered saline (TBS) buffer and blocked by 3% BSA. The blocked membranes were then incubated with any of anti-MCP-1, anti-p65, anti-P-p65, anti-IκBα, anti-P-IκBα, anti-IKKα/β, anti-P-IKKα/β or anti-β-actin primary antibodies (Abcam, Cambridge, MA, USA) diluted in the ratio of 1:1,000 for 2 h, in which antibody of β-actin was used as internal control. The primary antibodies were then washed with cold PBS buffer and were again incubated with HRP-conjugated secondary antibody (Abcam) diluted in the ratio of 1: 10,000 for 2 h. The signals on the membrane were then reacted with enhanced chemiluminescence (ECL, Thermo fisher scientific, Waltham, MA, USA) substrate for 1 min at room temperature. The signals on the membrane were detected through charge-coupled device (CCD, Thermo fisher scientific, USA) digital imaging. All of the experiments were triplicated.

Protein extraction from aortic tissue was done as previously described (Rivera-Torres, 2015 (link)). The cytosol and nuclear protein were obtained using Nuclear and Cytoplasmic Protein Extraction kit (CWBIO) according to the manufacturer’s instructions. Proteins were separated by SDS-PAGE and transferred electrophoretically to polyvinylidene fluoride membranes (Millipore). After blocking with 5% nonfat milk in TBST (pH 7.4, 20 mM Tris-HCl, 15 m NaCl, and 0.1% [vol/vol] Tween-20), the membranes were incubated overnight at 4°C with primary antibodies as follows: anti-TSH (bs-2676R) from Bioss; anti–NF-κB p65 (ab32536, clone E379), anti-IκBα (ab32518, E130) from Abcam; anti–p-IκBα (CST9246, clone 5A5), anti–p-Thr202/Tyr204 Erk1,2 (CST4695, clone 137F5), anti–t-ERK (CST4376, clone 20G11), anti–p-Thr180/Tyr182 p38MAPK (CST4511, clone D3F9), anti–t-p38 (CST9228, clone L53F8), anti–p-Thr183/Tyr185 JNK (CST4668 clone 81E11) from Cell Signaling Technology; anti–t-JNK (sc-7345, clone D-2) from Santa Cruz Biotechnology; and anti-GAPDH, (60004-1-ig, clone 1E6D9) and anti–laminin B (66095-1-ig, clone 3C10G12) from Proteintech. After washing with TBST, the membranes were incubated for 1 h at 25°C with the appropriate HRP-conjugated secondary antibody. The bands were visualized by FluorChemQ system using a chemiluminescence kit (Pierce).

In order to extract proteins, the cells were lysed in 1 mL of RIPA buffer supplemented with 1% PMSF and 1% phosphatase inhibitor, and the protein concentration was tested by using BCA kit (Beyotime Institute of Biotechnology, Shanghai, China). Then, in the use of 10% SDS-PAGE, 20 µg of the protein was separated and transferred to a PVDF membrane. Then, at room temperature, the membrane was blocked with 5% skim milk for 2 h and incubated at 4 °C overnight using the following primary antibodies: anti-PI3Kp85 (Cell Signaling Technology), anti-p-PI3K p85 (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), anti-p-Akt (Cell Signaling Technology), anti-NF-κBp65 (Cell Signaling Technology), anti-p-NF-ΚBp65 (Cell Signaling Technology), anti-P-IκBα (Abcam, MA, USA), and anti-GAPDH (Cell Signaling Technology). The membrane was incubated with HRP-labelled secondary antibodies at room temperature for 2 h, after three times washed with PBST. The membranes were washed again (three times) in use of PBST and visualized utilizing enhanced chemiluminescence.

The experimental procedure was performed as described in the previous study [32 (link)]. The total protein concentration of cells and tissues was measured using the BCA protein quantification method (Beyotime, China). Equal amounts of proteins were resolved by electrophoresis on 10% SDS gel and then transferred onto nitrocellulose membranes. Following blocked by incubation in 5% nonfat milk, membranes were probed with specific anti-CD9 (Cell Signaling Technology, 1:1000), anti- CD63 (Cell Signaling Technology, 1:1000), anti- CD81 (Cell Signaling Technology, 1:1000), anti- FGL1 (Abcam, 1:1500), anti-p-p65 (Abcam, 1:1500), anti-p65 (Abcam, 1:1000), anti-IκBα (Abcam, 1:1000), anti-p-IκBα (Abcam, 1:1500), anti-Bcl-2 (Abcam, 1:1500), anti-Bax (Abcam, 1:1500) and anti-cleaved-caspase 3 (Abcam, 1:1000) overnight at 4°C. After washing with Tris buffer saline (TBS) containing 0.24% Tween-20, membranes were then incubated for 60 min with horseradish peroxidase-conjugated secondary antibody. Then, an enhanced chemiluminescence system was used for visualization of protein signals, and the density of each protein band was analyzed using Image-Pro Plus6.0 (Media Cybernetics, Silver Spring, USA).

Total proteins were extracted from SUNE2 cells with the cell lysis buffer (RIPA; Beyotime, Shanghai, China). Samples were boiled for 10 min with 4× loading buffer (Takara, Shiga, Japan) and separated by 12% SDS-PAGE. Then, the proteins were transferred to polyvinylidine difluoride (PVDF) membranes (Sigma-Aldrich Co.). The membrane was blocked with 5% (wt/vol) skimmed milk powder and washed with Tris-buffered saline containing 0.1% Tween-20 (TBST); the PVDF membranes were then incubated with anti-β-actin (Abcam, Cambridge, UK), anti-p-IκBα (Abcam), anti-IκBα (Abcam), anti-p-p65 (Abcam), and anti-p65 (Abcam) overnight at 4°C, respectively. After three washes with TBST, the PVDF membranes were further probed with horseradish peroxidase-conjugated secondary antibodies (Stanta Cruz Biotechnology, Santa Cruz, CA, USA). Lastly, protein expression was quantified using VersaDoc 4000MP imaging system (Bio-Rad Laboratories Inc.).