Bca protein assay reagent

After the treatment of MCF-7 cells with compound 11/12a (0, 5, 10, and 20 μM) for 72 h, the total proteins of MCF-7 cells were extracted and their concentrations were balanced to the same level by BCA protein assay reagent (KGP902, KeyGen Biotech Co. Ltd., Nanjing, China), followed by 8 min of protein denaturation with SDS loading buffer at 100 °C. Proteins were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked with 5 mL of 5% fat-free dry milk in 1× Tris-buffered saline (TBS) containing 0.05% Tween 20 for 2 h. Then the membranes were incubated with Apaf-1 (Abcam, no: ab234436), Bax (Abcam, no: ab32503), Bcl-2 (Abcam, no: ab692), Bcl-xL (Abcam, no: ab32370), caspase-3 (Abcam, no: ab184787), caspase-8 (Abcam, no: ab25901), caspase-9 (Abcam, no: ab219590), cytochrome c (Abcam, no: ab133504), and β-actin (Abcam, no: ab8226) antibodies at 4 °C overnight, followed by treatment with secondary horseradish-peroxidase-conjugated anti-rabbitIgG (KeyGen Biotech Co. Ltd., no: KGAA35) for 2 h. Membranes were finally scanned in a ChemiDoc MP Imaging System (Bio-Rad) after 2 min incubation in Clarity Western ECL Substrate (Bio-Rad).

To identify sEV-BAT and sEV-WAT, equal amount of protein (20 μg) from adipocytes or sEVs were loaded for Western blot. To detect browning markers, equal amount of protein (20 μg) were extracted from treated cells at different times. Protein concentrations were measured using BCA Protein Assay Reagent (KeyGen BioTECH, KGP902, China). Samples were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred by electrophoresis to PVDF membranes (Millipore, Billerica, MA), blocked in 5% milk for at least 1 h and incubated with antigen-specific antibodies overnight at 4 °C. CD9 (220,642, Zen Bio, 1:1000), CD81 (381,296, Zen Bio, 1:1000), and Tumor susceptibility gene 101 protein (TSG101) (385,999, Zen Bio, 1:1000) were from Zen Bioscience; UCP1 (ab23841, Abcam, 1:1000) and ACTIN (ab3280, Abcam, 1:5000) were from Abcam; PGC-1α (D162041, BBI, 1:1000) were from BBI Life Sciences. Primary antibodies were then labeled for at least 1 h with horseradish peroxidase (HRP) conjugated secondary antibodies. Immunodetection was performed using High-sig ECL Western Blotting Substrate (Tanon,180–501) and was detected using a Bio-Rad Chemidoc imager.

Casepase-7, casepase-8, casepase-9, and Cyt C (mitochondrial cytochrome C) antibodies were purchased from Cell Signaling Technology. The antibody of p53 was purchased from Santa Cruz Biotechnology. Briefly, the SGC-7901 cells were assessed by the western blot analysis. Then, the total protein was extracted by chemical methods. Protein content was determined by BCA protein assay reagent (KeyGen, Nanjing, China). Equal amount of the proteins (50 μg) of each sample was resolved on SDS-PAGE and then electro transferred to PVDF membrane (Millipore Corp.). Nonspecific binding was blocked by incubation with 5 % nonfat milk in Tris-buffered saline containing 0.1 % Tween-20 (TBST) for 1 h at room temperature. The blots were probed with respective primary antibodies (antibodies used were against p53, casepase-7, casepase-8, casepase-9, Cyt C, and β-actin in 1:1000 dilutions) at room temperature overnight and washed three times with TBST. The blots were then incubated with secondary antibody for 30 min and washed again three times with TBST, and signals were visualized by chemiluminescent ECL detection system.