The total protein of HNSCC cells was extracted using SEMS lysis buffer (Beyotime, China); the protein contents were determined using a BCA protein assay kit (Beyotime, China). Equal amounts of proteins (40 μg) were separated using premade SDS-PAGE gels (Jinsirui, China) and transferred onto 0.22-μm polyvinylidene fluoride membranes (Merck Millipore, USA). The membranes were then incubated in 10% skim milk in PBS for 1 h at room temperature and incubated with the primary antibodies (1:1000) at 4°C overnight. The membranes were then probed with HRP-labeled rabbit antibodies (Beyotime, China) for 1 h, and an ECL (Beyotime, China) developing solution was added to enable the visualization of the protein bands, which were detected on a chemiluminometer (Amersham Imager 600). The antibodies against PLAU1, MMP1, MMP7, TIMP2, CDH1, and GAPDH were purchased from Abcam (UK).
Sems lysis buffer
Manufactured by Beyotime
Sourced in China
SEMS lysis buffer is a reagent used in the process of cell lysis. It is designed to disrupt the cell membrane and release the cellular contents, including proteins, nucleic acids, and other biomolecules, for further analysis or purification.
Automatically generated - may contain errors
2 protocols using sems lysis buffer
1
Protein Expression Analysis of HNSCC Cells
2
Western Blot Analysis of EZH2 and H3K27Me3
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Cells were collected at the indicated times in SEMS lysis buffer (Beyotime, China). The protein concentration was determined by a BCA protein assay kit (Beyotime, China). Equal amounts of proteins were separated using 10% polyacrylamide gels and transferred to 0.22-μm polyvinylidene fluoride (PVDF) membranes (Merck Millipore, USA). The membrane was soaked in 10% skim milk in PBS for 1 h at room temperature and incubated with primary antibody overnight at 4°C. Then, the membranes were probed with infrared (IR) dye-labeled secondary antibodies, and signals were detected using an Odyssey Infrared Imaging System (Biosciences, USA). GAPDH was used as a control. Antibodies (1:1,000) against EZH2, H3K27Me3, H3, and GAPDH were purchased from Cell Signaling Technology (Boston, MA, USA). The quantitative analysis of western blot was performed by ImageJ (NIH, Bethesda, MD, USA).
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