Bca kit

RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with 1% phenylmethylsulfonyl fluoride (PMSF, Dingguo, Beijing, China) was used for protein extraction of cells and tissues. The protein concentration was measured with a BCA kit (Dingguo, Beijing, China) according to the manufacturer’s instructions. Protein samples (40 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with TBST containing 5% skimmed milk at room temperature for 1.5 hours and incubated with specific primary antibody diluted with 5% BSA at 4°C overnight. After incubation with secondary antibody for 2 hours at room temperature, the protein signals were detected using a ECL chemiluminescence kit (Advansta, San Jose, CA, USA). Immunoprecipitation was performed as previously described (34 (link)). Grayscale value analysis of all protein bands was performed via ImageJ 1.46r software (NIH, Bethesda, MD, USA).

Cells were homogenated in RIPA (Santa Cruz Technology, Santa Cruz, CA, USA). After centrifugation at 12,000× g for 20 min at 4 °C, protein concentrations in the supernatants were measured by a BCA kit (Dingguo, Changchun, China). Denatured proteins were separated on 10%–12% SDS-PAGE gels and transferred to 0.2 μm nitrocellulose membrane. Blocking in TBS containing 5% non-fat milk and 0.5% BSA for 1 h at room temperature was followed by incubation with primary antibodies against Bcl-2 (Santa Cruz Biotechnology), Bax (Santa Cruz Biotechnology), cleaved caspase-3 (Abcam, Cambridge, UK), or p21 (Abcam) overnight at 4 °C, and then with a secondary antibody for 1 h at room temperature. Signals were revealed using an ECL kit (Bio-Rad Laboratories).

HT-29 and H1299 cells were grown in their respective media supplemented with 10% exosome-free FBS, in which the exosomes were removed from the FBS by ultracentrifugation. The culture medium was centrifuged at 2000 g for 20 min and filtered through a 0.22 μm filter to remove cell debris. Thereafter, the exosomes were extracted from the filtered media using the Total Exosome Isolation Reagent (Cat. 4478359, Thermo Fisher Scientific), according to the manufacturer’s instructions. The harvested pellet was then lysed using IP lysis buffer, and the protein quantity in the exosomes was measured using a bicinchoninic acid assay (BCA) kit (Dingguo, Guangzhou, China).
Ultracentrifugation was conducted as follows: the filtered medium was first centrifuged at 100,000 g for 80 min at 4 °C in an ultracentrifuge. Thereafter, the pellet was resuspended in PBS and centrifuged again for 80 min at 4 °C. After centrifugation, the pellet was dissolved in cold PBS and the protein concentration was quantity using the BCA kit. The exosome samples were stored at −80 °C. CD63, CD9, CD81, Alix, and TSG101 (Proteintech, Wuhan, China) were considered as the positive markers, while Calnexin (sigma) was considered as a negative marker for exosomes for the WB analysis. Furthermore, the isolated exosomes were subjected to NTA analysis and observed under TEM (APTBIO, Shanghai, China).

E. coli JM109 served as the host strain for site-specific mutagenesis. E. coli Rosetta gami-B (DE3) (Weidi Biotech, Shanghai, China) was used to express various mutant enzymes. The gene for BbPETase^CD was synthesized by Genewiz (Suzhou, China) and using pET-22b(+) plasmid as gene vector. DNA sequencing was performed by Genewiz (Suzhou, China). 5 mL His Trap^TM FF affinity chromatography column was purchased from GE Healthcare (9 × 15.7 mm, 90 μm, Marlborough, MA, USA). Fast pfu enzyme and DpnI enzyme were purchased from TransGen Biotech (Beijing, China). A plasmid extraction kit was purchased from Beyotime (Shanghai, China). A BCA kit was purchased from Dingguo Biotech (Tianjin, China). A sulfhydryl assay kit was purchased from Bestbio (Shanghai, China). The crystallinity of PET films was measured using DSC by Standard (Qingdao, China). Other reagents were purchased from Sangon Biotech (Shanghai, China), Heowns (Tianjin, China), or Meryer (Shanghai, China).