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5 protocols using phospho iκbα ser32 36 5a5

1

Western Blot Analysis of Protein Expression

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After 24 h post-transfection, cells were collected, and protein extracts obtained by adding Leammli sample buffer (Bio Rad, Ca, USA). Equal amounts of proteins were loaded and separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked using 7.5% milk in TBS/0.01% Tween buffer and then incubated with the indicated antibodies. Primary antibodies were prepared in TBS/0.01% Tween buffer as follows: anti-GAPDH (1:1000) (32233; Santa Cruz Biotechnology, Santa Cruz, California), anti-HA (1:1000) (C29F4; Cell Signaling Technology Europe, B.V), anti-p52NFκB (1:1000) (298; Santa Cruz Biotechnology, Santa Cruz, California), α-Actinin (1:1000) (17829; Santa Cruz Biotechnology, Santa Cruz, California) and Phospho-IκBα (Ser32/36)(5A5) (9246; Cell signaling Technology Europe, B.V) Membranes were washed three times with TBS/0.01% Tween buffer and incubated with HRP coupled secondary anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, California). Finally, Proteins were visualized using the ChemoLuminiscent Reagent (Merck Millipore, Burlington, Massachusetts, U.S) according to the manufacturer’s instructions.
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2

Immunohistochemistry Staining of Aorta

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The procedure guidance for staining of immunohistochemistry was according to previous study with some modifications as explained by the manufacturer's (DAKO, Agilent, USA) instructions.31 (link) After that, the ∼5  μm paraffin-fixed thoracic aorta sections underwent orderly incubation with an antigen retrieval solution (Sigma-Aldrich, USA), and with a primary antibody (iNOS; ab15323, Abcam, USA, or phospho-IκBα (Ser32/36; 5A5; Cell Signaling, USA), or IκBα (Cell Signaling, USA), or NOX1 (NOX1 (H-75; Santa Cruz Biotech, USA), or NOX4 (Santa Cruz Biotech, USA). All primary antibodies were at 1:100, and each slide was incubated for 18 h at 4 °C. Non-immunized goat serum were used as the primary antibodies for negative controls. Finally, the immunohistochemistry staining results were photographed with a Nikon Eclipse TE 2000-S (Tokyo, Japan). For image process and analysis, the microscope was equipped with Nikon DS Fi1 digital camera and NIS-Element F software.
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Immunoblotting Antibodies for Cell Signaling

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Primary antibodies reactive to β-actin (AC-15; Sigma–Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma–Aldrich, St. Louis, MO, USA), ICAM-1 (15.2; Leinco Technologies, St. Louis, MO, USA), FLIP (Dave-2; Alexis® Biochemicals, San Diego, CA, USA), IκBα (25; BD Biosciences, San Jose, CA, USA), Mcl-1 (D35A5; Cell Signaling Technology, Danvers, MA, USA), NF-κB p50 (H-119; Santa Cruz Biotechnology, Dallas, TX, USA), NF-κB p65 (C-20; Santa Cruz Biotechnology, Dallas, TX, USA), Poly(ADP-ribose) polymerase (PARP) (C2-10; Trevigen, Gaithersburg, MD, USA), phospho-IκBα (Ser32/36) (5A5; Cell Signaling Technology, Danvers, MA, USA), and phospho-NF-κB p65 (Ser536) (93H1; Cell Signaling Technology, Danvers, MA, USA) were obtained.
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4

Immunoblot Analysis of Cell Signaling

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Immunoblot protein analysis was performed according to standard protocols. Antibodies used included anti–human α-SMA (1A4; Dako), p16 (DCS-50; Abcam), STAT-1 (1/Stat1; BD), phospho–STAT-1 (Tyr701), NF-κB p65 (C22B4), phospho-IκBα (Ser32/36; 5A5; Cell Signaling Technology), anti-IκBα (6A920), and histone 2B (FL-126; Santa Cruz Biotechnology, Inc.).
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5

Murine Macrophage-like Cell Signaling

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Murine macrophage-like RAW264.7 cells were obtained from the European Collection of Authenticated Cell Cultures (Salisbury, UK) . Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA) . RPMI 1640 medium, penicillin/streptomycin, and β-carotene were purchased from FUJIFILM Wako Pure Chemical Co., Ltd. (Osaka, Japan) . Primary antibodies targeting phospho-SAPK/Jun N-terminal kinase (JNK) ( Thr183/Tyr185; rabbit polyclonal antibody) , SAPK/JNK (rabbit polyclonal antibody) , NF-κ B p65 (D14E12; rabbit monoclonal antibody) , and phospho-Iκ Bα (Ser32/36; 5A5; mouse monoclonal antibody) were purchased from Cell Signaling Technology (Beverly, MA, USA
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