Goat anti mouse igg

Proteins were extracted with SDS lysis buffer and 1 × protease inhibitor cocktail (Roche, Germany), and the concentrations were measured by Enhanced BCA Protein assay kit (Thermo Fisher Scientific, USA). Proteins were separated by SDS-PAGE, then transferred onto a PVDF membrane (Millipore, USA), followed by blocking with 5% milk (BD, USA) in TBST (Tris‐buffered saline with Tween‐20) solution for 1.5 h at room temperature. The membrane was incubated at 4°C overnight with corresponding primary antibodies to C1QBP (Abcam, ab101267; 1:2000), XDH (Affinity Biosciences, DF8111; 1:1000), caspase-3 (Abcam, ab184787; 1:2000), bcl2 (Abcam, ab182858; 1:2000), bax (Abcam, ab32503; 1:2000) and β-actin (Affinity Biosciences, T0022; 1:3000). After washing three times in TBST, the membranes were further incubated with HRP‐conjugated goat anti‐rabbit IgG (Affinity Biosciences, S0001; 1:5000) or goat anti‐mouse IgG (Affinity Biosciences, S0002; 1:4000) secondary antibodies for 1 h at room temperature and developed by ECL Western Blotting Detection Reagents (Millipore, USA). The expression of target protein was normalized with β-actin.

Proteins were extracted from breast cancer tissues or cells using RIPA lysis buffer supplemented with protease inhibitor. Next, total proteins were separated using gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime) via electroblotting. The PVDF membranes were blocked with 5% skim milk, after which they were incubated with primary antibodies rabbit anti‐Pygo2 (1:1000, Abcam), rabbit anti‐E‐cadherin (1:1000, CST), rabbit anti‐vimentin (1:2000, Abcam), rabbit anti‐β‐catenin (1:1000, Abcam), rabbit anti‐c‐Myc (1:1000, Abcam), rabbit anti‐cyclin D1 (1:1000, Abcam) and rabbit anti‐GAPDH (1:4000, Santa Cruz) overnight at 4°C. Subsequently, the membranes were incubated with the appropriate secondary antibodies, including goat antimouse IgG (1:10 000, Affinity) and goat anti‐rabbit IgG (1:10 000, Affinity), for 1 hour. ECL detection reagent (Santa Cruz) was added on the membranes to detect signals. GAPDH used as a loading control. The greyscale values of protein bands were analysed using ImageJ.