Western Blot Analysis of Protein Targets

Total cell protein was prepared using RIPA Lysis Buffer (Beyotime Institute of Biotechnology). Protein was measured using a BCA protein Assay Kit (CWBIO). Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes using a Bio-Rad Mini PROTEAN 3 system (Bio-Rad Laboratories, Inc.). The membranes were blocked with PBS containing 5% milk and 0.1% Tween-20 at room temperature for 1 h. The primary antibodies were as follows: β-actin (mouse monoclonal, dilution 1:10,000; A4551) was from Sigma-Aldrich; Merck KGaA. Lamin A (mouse monoclonal, dilution 1:1,000; sc-71481) was obtained from Santa Cruz Biotechnology, Inc. Anti-NF-κB p65 (rabbit polyclonal, dilution 1:1,000; ab16502), anti-Histone H3 acetyl K9 (rabbit polyclonal, dilution 1:5,000, ab4441), anti-HDAC1(rabbit polyclonal, dilution 1:5,000, ab109411) and anti-IκB-α (rabbit polyclonal, dilution 1:5,000, ab32518) were purchased from Abcam. Horseradish peroxidase-conjugated anti-mouse (1:2,500 dilution) or anti-rabbit (1:2,500 dilution) secondary antibodies were purchased from Bioworld Technology, Inc. Immunoreactive bands were visualized by using the Amersham ECL Western Blotting Detection Kit (Cytiva) according to the manufacturer's instructions. β-actin served as a loading control (19 (link),23 (link)).

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Lin S., Tian C., Li J., Liu B., Ma T., Chen K., Gong W., Wang J.M, & Huang J. (2021). Differential MUC22 expression by epigenetic alterations in human lung squamous cell carcinoma and adenocarcinoma. Oncology Reports, 45(5), 78.

Publication 2021

RIPA Lysis Buffer BCA protein Assay Kit Bio-Rad Mini PROTEAN 3 system β-actin Lamin A Anti-NF-κB p65 anti-Histone H3 acetyl K9 anti-HDAC1 anti-IκB-α Amersham ECL Western Blotting Detection Kit

Actin Antibodies Assay Buffer Cell Dilution Histone h3 Horseradish peroxidase Iκb α Lamin a Membranes Milk Mouse Nf κb p65 Proteins Pvdf Rabbit Ripa Sds page Tween 20

Corresponding Organization :

Other organizations : Beijing Chest Hospital, Capital Medical University, Xuchang University, Henan University of Science and Technology, National Cancer Institute, Frederick National Laboratory for Cancer Research, Center for Cancer Research, Leidos (United States)

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of β-actin, Lamin A, NF-κB p65, Histone H3 acetyl K9, HDAC1, and IκB-α

control variables

Total cell protein was prepared using RIPA Lysis Buffer (Beyotime Institute of Biotechnology)
Protein was measured using a BCA protein Assay Kit (CWBIO)
Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes using a Bio-Rad Mini PROTEAN 3 system (Bio-Rad Laboratories, Inc.)
The membranes were blocked with PBS containing 5% milk and 0.1% Tween-20 at room temperature for 1 h
Horseradish peroxidase-conjugated anti-mouse (1:2,500 dilution) or anti-rabbit (1:2,500 dilution) secondary antibodies were used
β-actin served as a loading control

controls

Positive control: β-actin
Negative control: None explicitly mentioned

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