Dansylcadaverine

B. amyloliquefaciens TCCC 111018, B. cereus TCCC 111006, B. safensis TCCC 111022, and B. aryabhattai TCCC 11368 were kept in our laboratory, and they were incubated at 37 °C in liquid LB medium. Escherichia coli BL21 (DE3) was utilized for expressing the recombinant enzyme, and kanamycin was added when necessary. The pET-28a(+) was employed as an expression vector.
Reagents were provided by the following sources: N,N-dimethylcasein and dansyl cadaverine [N-(5-aminopentyl)-5-dimethylamino-1-naphthalenesulfonamide, MDC] were ordered from Sigma-Aldrich Co. (St. Louis, MO, USA) and Aladdin Chemical Co., Ltd. (Shanghai, China), respectively. Bovine serum albumin (BSA, ≥98%), soy protein isolate (SPI, ≥88%), and whey protein (WP, ≥80%) were provided by Solarbio Science & Technology Co., Ltd. (Beijing, China), Macklin Biochemical Co., Ltd. (Shanghai, China), and Sigma-Aldrich (St Louis, MO, USA), respectively.

Cadmium acetate (CdAc₂), chloroquine (CQ), rapamycin (RAP), dansylcadaverine (MDC), anti-LC3 (Lot#: 065M4757V) and Hoechst 33258 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences (San Diego, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Trypsin was obtained from Amresco (Solon, OH, USA). Antibodies against Bax (Ref. No. : 03/2013), Bcl-2 (Ref. No. : 01/2013), cleaved-PARP (Ref. No. : 12/2012), autophagy gene 5 (Atg5) (Ref. No. : 01/2013), β-actin (Ref. No.: 06/2012) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (Ref. No. : 10/2012) were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-beclin 1 (Lot#: J0112), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescence (ECL) solution was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Other chemicals and reagents were purchased locally and were all at analytical grade.

Ethacrynic acid was obtained from Sigma Aldrich (St. Louis, MO). Human plasma thrombin, α₂-antiplasmin, and FXIIIa were obtained from Haematologic Technologies (Essex Junction, VT). N,N-dimethyl-casein, dansyl-cadaverine, and dithiothreitol (DTT) were also from Sigma Aldrich. Dabigatran, rivaroxaban, and AntiF11 (mouse monoclonal antibody from Abnova™) for plasma studies as well as Coomassie Brilliant Blue for gel electrophoresis were from Fisher Scientific (Waltham, MA). Fibrinogen was from Haematologic Technologies. Stock solution of FXIIIa was prepared in 50 mM Tris–HCl, 1 mM CaCl₂, 100 mM NaCl, 0.1% PEG8000, 0.02% Tween80, and 2 mg/mL N,N–dimethylcasein. For the clotting assays, pooled normal human plasma was purchased from George King Bio-Medical (Overland Park, KS). APTT reagent containing ellagic acid, thromboplastin‒D (PT reagent), and 25 mM solution of CaCl₂ were purchased from Thermo Fisher Scientific. All experiments in this paper were repeated at least two times. For molecular modeling studies, initial structure of FXIIIa (4kty.pdb) was prepared by removing the crystallographic water molecules and adding hydrogen atoms consistent with the physiologic pH of 7 using Maestro 12.4.1. Docking studies were carried out by generating a non-covalent pose using Glide (Schrodinger Suite 2020), and then, by using the covalent docking program (Schrodinger Suite 2020).