Ni2 affinity chromatography column

An RNAprep pure Tissue Kit (Tiangen, Beijing, China) was used to extract total RNA from PSCs, and cDNA was subsequently synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Specific primers (Additional file 1: Table S1) were designed based on the full-length coding sequences of the four EgANXBs. After PCR, the products were extracted from the gel and cloned into vector pMD19-T (Takara, Dalian, China) for sequencing. Then, the pMD19-T-EgANXBs plasmids were subcloned into vector pET-32a ( +) (Invitrogen, Waltham, MA, USA) and transformed into Escherichia coli BL21 or Rosetta (DE3) cells (Tiangen), followed by induction with 0.24 mg/ml isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 ℃ for 12 h. The recombinant (r)EgANXBs were purified using a Ni²⁺ affinity chromatography column (Bio-Rad, Hercules, CA, USA). The imidazole was removed, and the concentration of each protein was determined using a bicinchoninic acid (BCA) protein quantification kit (Bestbio). The purified proteins (0.5 mg/well) were assessed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the gels were stained with coomassie blue.

The correctly sequenced EgAnxB3 and EgAnxB38 plasmids were digested with restriction enzymes, ligated into the pET32a(+) plasmid. The resulting recombinant plasmids were transformed into Escherichia coli BL21 (DE3) (Tiangen, Beijing). E. coli cells containing pET32a-EgAnxB3 and pET32a-EgAnxB38 were cultivated at 37 °C for 8 h. Then the transformants were induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. The recombinant proteins were purified using a Ni²⁺ affinity chromatography column (Bio-Rad, Hercules, CA).

Total RNA was extracted from PSCs using the RNAprep pure Tissue Kit (Tiangen, China) according to the manufacturer’s guidelines, and the Thermo Scientific RevertAid (Thermo Fisher Scientific, USA) was used to synthesize first-strand cDNA. The sequences of EgE2D2 and EgE2N were amplified with primers described in Table 1. Purified polymerase chain reaction (PCR) products were cloned into a pET32a (+) vector and transformed into E. coli BL21 (DE3). The BL21 strain was cultured at 37 °C, 160 rpm for 6 h, and then added with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce protein expression. Ni²⁺ affinity chromatography column (Bio-Rad, USA) was used to purify the recombinant proteins.Table 1

Primer sequences

Gene	Forward primer (5′-3′)	Reverse primer (5′-3′)
EgE2D2	CGGATCCATGGCCCTGAAGAGGATTC	CGGAATTCCTACATTGCGTACTTCTGAGTCC
EgE2N	CGAGCTCATGAGTGGACATCTTCCTACAC	CCTCGAGCTAGCGGAAGTCGGAAG
α-SMA	GACAGCTACGTGGGTGACGAA	TTTTCCATGTCGTCCCAGTTG
COL1A1	GTGCGATGACGTGATCTGTGA	CGGTGGTTTCTTGGTCGGT
COL1A2	GTTGCTGCTTGCAGTAACCTT	AGGGCCAAGTCCAACTCCTT
TIMP1	TGCAGGATGGACTCTTGCAC	GCATTCCTCACAGCCAACAG
GAPDH	CAAGGTCATCCATGACAACTTTG	GTCCACCACCCTGTTGCTGTAG

Black and bold italics signify restriction enzyme sites