Enhanced chemiluminescence kit

Cells were washed three times with cold PBS and total cellular proteins were extracted using a modified radioimmunoprecipitation assay buffer (OriGene Technologies, Inc., Beijing, China) with 1 mM phenylmethane sulfonyl fluoride for 30 min. A bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) was used to measure the protein concentration. Equal amounts of protein (30 µg/lane) were separated via 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk at room temperature for 1.5 h, followed by incubation overnight at 4°C with the following primary antibodies against: HLA-G (1:1,000; ab135736; Abcam, Cambridge, UK), MMP7 (1:1,000; cat. no. 3801; Cell Signaling Technology, Inc., Danvers, MA, USA); β-catenin (1:1,000; cat. no. 8480, Cell Signaling Technology, Inc.); TCF-4 (1:1,000; cat. no. 2565; Cell Signaling Technology, Inc.) and GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.). The membranes were subsequently incubated for 2 h at room temperature with an anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.). Protein bands were visualized using an enhanced chemiluminescence kit (Applygen Technologies, Inc., Beijing, China). GAPDH was used as an internal control.

Total protein was extracted from cells using the radioimmunoprecipitation assay buffer kit (Thermo Fisher Scientific, Inc.). Protein concentration was quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.) prior to separation via SDS-PAGE on a 10% gel at 30 µg protein/lane. The separated proteins were then transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 1 h. Following blocking, the membranes were then incubated with primary antibodies against Bcl-2 (cat no. 4223), Bax (cat no. 5023) and β-actin (cat no. 4970; all dilution: 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. The membranes were washed with phosphate buffer saline (PBS)-0.05% Tween 20 5 times and then incubated with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody (cat no. 7074; dilution: 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. The protein bands were visualized using an enhanced chemiluminescence kit (Applygen Technologies, Inc.) according to manufacturer's protocols. Densitometry was performed using the ImageJ software (version 1.38X; National Institutes of Health).

The cells were washed in 1X PBS and lysed in lysis buffer as described (13 (link)). The total protein concentrations was determined using the Bradford method. The total protein or protein fractions (50 µg/lane) were loaded and separated by 10 or 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk powder for 45 min at room temperature. The membranes were then incubated with primary antibodies directed against β-actin (dilution 1:2,000), Bax (dilution 1:1,000), Bcl-2 (dilution 1:1,000), caspase-3 (dilution 1:1,000), caspase-9 (dilution 1:1,000), cytochrome c dilution 1:1,000), p70S6K (dilution 1:500), phospho-p70 S6K (Thr389) (dilution 1:1,000), MAP4K3 rabbit Ab (dilution 1:500), myc (dilution 1:2,000), thiophosphate ester (dilution 1:5,000), LC3 (dilution 1:1,000) and P62 (dilution 1:1,000) overnight at 4°C. The membranes were subsequently washed three times with PBS-0.1% Tween-20 for 10 min and were then incubated with goat anti-mouse (dilution 1:10,000) or goat anti-rabbit (dilution 1:5,000) secondary antibodies for 1 h at room temperature. The expression of individual proteins was detected with an enhanced chemiluminescence kit (Applygen Technologies, Inc., Beijing, China). The densitometric values of the bands were measured using ImageQuant TL software (version 8.1; GE Life Sciences, Chicago, IL, USA).