Anti rabbit igg

After different treatments, the medium was aspirated and SiHa cells were lysed with ice-cold RIPA lysis buffer. Protein concentrations were determined using the bicinchoninic acid method. After adjustment to a similar level of total protein concentration, samples were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in TBST buffer (20 mM Tris–HCl, 137 mM NaCl, and 0.1% Tween 20, pH 8.0) for 1 hour at room temperature prior to incubation with specific antibodies to cyclin E, p27, Skp2, and GADPH overnight at 4°C. After washing and reaction with horseradish peroxidase-conjugated anti-mouse IgG (Beijing Zhong Shan Golden Bridge Biological Technology Co. Ltd., Beijing, People’s Republic of China), or anti-rabbit IgG (Beyotime, People’s Republic of China) secondary antibodies for 1 hour, the membranes were washed with TBST buffer three times and the proteins on the membrane were detected using an enhanced chemiluminescence substrate (Beyotime).

U251MG cells (1×10⁵) were treated with MCL (0, 5, 10 and 15 µM) for 48 h at 37°C, and then total protein was extracted by RIPA lysis buffer and quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Protein (30–50 µg) was separated via 10–12% SDS-PAGE. The separated proteins were subsequently transferred onto a PVDF membrane and blocked with 5% non-fat dry milk (Beyotime Institute of Biotechnology) for 1h at room temperature. The membranes were then incubated at 4°C overnight with the following primary antibodies: Anti-cleaved caspase-3 (1:1,000), anti-cleaved caspase-9 (1:1,000), anti-COX-2 (1:1,000), anti-p-IκBα (1:500), anti-IκBα (1:1,000), anti-β-actin (1:2,000), anti-Bcl-2 (1:2,000), anti-Bax (1:2,000), anti-Vimentin (1:1,500), anti-MMP-9 (1:800) and anti-N-cadherin (1:500). Membranes were washed three times with 1X Tris-buffered saline with 0.1% Tween (Beyotime Institute of Biotechnology) and incubated with the anti-rabbit IgG (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) or anti-mouse IgG (1:5,000; cat. no. 7076; Cell Signaling Technology, Inc.) secondary antibodies for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology) and semi-quantification was performed using ImageQuant TL 7.0 software (GE Healthcare).

IF assay was performed via incubating 5-micron-thick sections in xylene for 15 min. The sections were dehydrated in ethanol (75 and 85%) for 5 min and washed in dH₂O; immersed in EDTA antigen retrieval buffer (pH 8.0); and maintained at boiling temperature for 7 to 8 min. To prevent the buffer solution from evaporation, the samples were cooled in PBS (pH 8.0) for 5 min using a rocker device. Liquid blocking pens were used to stop liquid elimination. After being incubated in spontaneous fluorescence quenching reagent for 5 min and being washed in fresh water, they were again incubated with primary antibody (diluted with PBS) goat-serum at room temperature and then with anti-CD8α antibodies (1:500 Biomos; Technology Co., Ltd., Beijing, China) in blocking solution at 4°C overnight, and then again incubated with FITC (1:400 Services-bio Co., Ltd., Wuhan, China) labeled antirabbit igG and mounted with DAPI (Beyotime Biotechnology, Co., Ltd., Jiangsu, China). Finally, the slides were observed under an inverted microscope (Nikon TE2000, Tokyo, Japan).