Peroxidase conjugated goat anti rabbit igg h l secondary antibody

The proteins of the parental adherent and tumor sphere cells were prepared with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), separated by 12% SDS-PAGE, transferred onto PVDF membranes using a semi-dry blotting apparatus (Bio-Rad, Hercules, CA, USA), and blocked in 5% non-fat milk. The membranes were subsequently incubated with the corresponding primary antibodies, as indicated: a rabbit anti-β-actin (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) diluted 1:300, rabbit anti-Oct4 and rabbit anti-Sox2 (BioLegend, San Diego, CA) diluted 1:1,000. Antibody recognition was detected with peroxidase-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China) used at 1:3,000 dilutions. Antibody-bound proteins were detected with a BeyoECL Plus kit (Beyotime Institute of Biotechnology) and western blotting analysis system (Universal Hood II, Bio-Rad, USA ).

Both morusin treated breast cancer cells and tumor tissues were lysated with RIPA lysis buffer, centrifuged, the supernatants were collected and quantified with UV spectrophotometer. Samples containing 30 μg protein were mixed with loading buffer (5×), boiled for 5 min, separated by 12 % SDS-PAGE, transferred PVDF membranes using a semi-dry blotting apparatus (Bio-Rad, Hercules, CA, USA), and then blocked in 5 % non-fat milk at RT for 1 h. The PVDF membranes were incubated with primary antibodies of rabbit anti-C/EBP β,rabbit anti-PPARγ, rabbit anti-adipsin D,rabbit anti- perilipin A + B, rabbit anti-Bcl-2, rabbit anti-Bax, rabbit anti-active caspase-3 and rabbit anti-β-actin (Beijing Biosynthesis Biotechnology Co., LTD, Beijing, China) diluted 1:300. Antibody recognition was detected with peroxidase-conjugated goat anti-rabbit IgG (H + L) secondary antibody (Zhongshan Goldenbridge Biotechnology Co., LTD, Beijing, China) used at 1:6000 dilutions, antibody-bound proteins were detected by Chemiluminescent HRP Substrate (Milipore Corporation, Billera, USA) and western blotting analysis system (Universal Hood II, Bio-Rad, USA), and normalized to β-actin and semi-quantified using the ChemiDocTM XRS (Bio-Rad, USA).

Cells or tumor tissues were collected and lysed using RIPA lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Beyotime Biotechnology). Cell lysates in 1× loading buffer were resolved by 12% SDS-PAGE and transferred on polyvinylidene fluoride membranes (Milipore, Bedford, MA, USA). After blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary rabbit antibodies against β-actin, Sox2, Oct4, ALDH1, Nanog, CD49f (1:300, Beijing Biosynthesis Biotechnology Co., LTD, Beijing, China), Vimentin, E-cadherin, and N-cadherin (1:1000, Proteintech Group, Chicago, USA) overnight at 4 °C. The primary antibodies were detected with peroxidase-conjugated goat anti-rabbit IgG (H + L) secondary antibody (Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China). Positive signals were detected using the BeyoECL Plus kit (Beyotime Institute of Biotechnology, Shanghai, China) and a western blot analysis system (Universal Hood II, Bio-Rad, USA).