Mouse anti b2m

Mouse anti-MMP2, mouse anti-PP2Ac, and mouse anti-B2M antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Total protein was extracted using a lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and supplemented with protease inhibitor cocktail kit (Roche). The protein extract was loaded onto an SDS-polyacrylamide gel, size-fractionated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, CA, USA). After blocking in 5% non-fat milk for 1 hour, the membranes were incubated overnight with primary antibodies at 4 °C. The protein expression was determined using horseradish peroxidase-conjugated antibodies followed by enhanced chemiluminescence (ECL, Millipore, St Charles, MO, USA) detection. The intensity of the bands was captured by JS-1035 image analysis scanning system (Peiqing Science & Technology, Shanghai, China). B2M was used as the internal control.

Rabbit anti-CDK1 was purchased from Cell Signaling Technology (MA, USA). Rabbit anti-p21, mouse anti-PP2Ac, and mouse anti-B2M antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Total protein was extracted using a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, supplemented with protease inhibitors (10 mg/mL leupeptin, 10 mg/mL aprotinin, 10 mg/mL pepstatin A, and 1 mM 4-[2-aminoethyl] benzenesulfonyl fluoride). The protein extract was loaded onto an SDS-polyacrylamide gel, size-fractionated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight with primary antibodies at 4°C. The protein expression was determined using horseradish peroxidase-conjugated antibodies followed by enhanced chemiluminescence (ECL, Millipore, St Charles, MO, USA) detection. The intensity of the bands was captured by JS-1035 image analysis scanning system (Peiqing Science & Technology, Shanghai, China). B2M was used as the internal control.

Whole cell proteins were isolated using mammalian protein extraction reagent M-PER (Invitrogen) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were determined using the Bradford assay (Bio-Rad, CA, USA). Equal amounts of protein samples (10 μg) were loaded onto NuPAGE™ 4–12% SDS-PAGE gels (Invitrogen), electrophoresed, and transferred to polyvinylidene fluoride membranes (Invitrogen). The membranes were subsequently incubated with primary antibodies overnight at 4 °C followed by secondary antibodies for 1 h at room temperature. Mouse anti-HLA-E monoclonal antibody (Acris, Rockville, MD, USA), mouse anti-B2M, or anti-β-actin antibody (Santa Cruz, TX, USA) were used as primary antibodies and goat anti-mouse IgG-horseradish peroxidase (HRP) antibody (Santa Cruz) was used as the secondary antibody. The immunostained blots were visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) and the signal detected using an EZ-Capture II chemiluninescence charge-coupled device (CCD) imaging system (ATTO, Amherst, NY, USA).