Enhanced chemiluminescence western blotting substrate kit

Transfected SK-BR-3 and BT-549 cells were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Proteins (50 µg per lane) were separated via SDS-PAGE on a 10% gel then transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Inc.). The membranes were blocked with 5% dry milk in Tris-buffered saline (TBS) with 0.2% of Tween-20 at 4°C overnight. The membranes were subsequently incubated with primary antibodies against E-cadherin (1:500; ab15148; Abcam), N-cadherin (1:500; ab18203; Abcam), Vimentin (1:500; ab8978; Abcam), and GAPDH (1:250; ab9485; Abcam) at room temperature for 3 h. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; ab6721; Abcam) at room temperature for 1 h. The protein signals were detected using an Enhanced Chemiluminescence Western Blotting Substrate kit (Thermo Fisher Scientific, Inc.) and quantified using ImageJ software v1.46 (National Institutes of Health). GAPDH was used as an internal control.

For protein extraction, cells were lysed in RIPA buffer (Beyotime Biotechnology) supplemented with protease inhibitors (Beyotime Institute of Biotechnology). After incubation on ice for 15 min, the lysate was centrifuged at 13,800 × g for 15 min at 4°C, and the supernatant was collected. Protein concentration was calculated using a BCA Protein Assay kit (cat. no. P0010S; Beyotime Institute of Biotechnology). Protein extracts were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk and incubated with rabbit polyclonal anti-intraflagellar transport 57 (IFT57) (1:1,000 dilution; cat. no. ab5205; Abcam) and rabbit anti-GAPDH (1:1,000 dilution; cat. no. 2118; Cell Signaling Technology, Inc.) overnight at 4°C, the membranes were incubated with anti-rabbit IgG (1:5,000 dilution; cat. no. AQ132P; EMD Millipore). After washing with TBS-Tween-20, the protein bands were detected using an enhanced chemiluminescence western blotting substrate kit (Thermo Fisher Scientific, Inc.). Relative protein levels were quantified by scanning densitometry using ImageJ software (version 1.4.3; National Institutes of Health).

Western blotting was used to identify and semiquantitative analysis of targeted protein. β-actin was utilized as an endogenous reference. Briefly, samples were extracted and separated by SDS-polyacrylamide gel electrophoresis (PAGE). Then, the samples were transferred onto polyvinylidene fluoride (PVDF) membranes to identify and semiquantitate targeted protein expression. The specific primary antibodies (cleaved PRRP, cleaved caspase3, CELF2, PRR11, p-PI3 K, p-AKT, p65-NF-κB, Bcl2, cleaved caspase9, cyclin D, E-cadherin, N-cadherin, vimentin, and β-actin) were all purchased from Cell Signaling Technology (CST) company (Boston, MA, USA). The specific primary antibodies were diluted to the appropriate concentration and incubated for 1 h at 37 °C, and then washed 3 times with PBS and incubated with corresponding secondary antibody (CST, Boston, MA, USA) for 1 h at 37 °C. Finally, the bands of membranes were detected using enhanced chemiluminescence Western blotting substrate kit (Thermo Fisher Scientific).