2.2. Chemicals, Antibodies and Plasmids

(20S)G-Rh2, etoposide, phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich.

Antibodies for Twist (25465-1-AP), SIP1 (21672-1-AP), Slug (12129-1-AP), Snail 1 (13099-1-AP), MMP-2 (10373-2-AP), MMP-9 (10375-2-AP), Anxa2 (11256-1-AP), β-actin (66009-1-lg) and myc-tag (16286-1-AP, 60003-2-lg) were purchased from Proteintech (Proteintech Group, Inc.). Antibodies for E-cadherin (sc-8426), N-cadherin (sc-59987), NF-κB p50 subunit (sc-7178), Anxa2 (sc-47696) and IgG (sc-2025) were purchased from Santa Cruz Biotechnology. HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (31460) and HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (31430) were purchased from Invitrogen.

Plasmids for over-expression of wild-type Anxa2 (pcs4-Anxa2-WT-myc) and Anxa2 K031A mutant (pcs4-Anxa2-K301A-myc) were shown as described [28]. A truncation with 1–33 deletion of Anxa2 was amplified by polymerase chain reaction (PCR), followed by a recombination into pcs4-myc vector for over-expression of Anxa2-delta N-terminus (pcs4-Anxa2-dN-myc). For lentivirus package, pLVX-TetOne-Puro (Clontech, 631849), pMD2.G (Addgene plasmid # 12259) and psPAX2 (Addgene plasmid # 12260) were gifts from Professor Zhihua Zou (Collage of Life Sciences, Jilin University). C-terminus-Myc-tagged Anxa2 full-length protein as well as K301A mutant and delta-N truncation were amplified from pcs4 vector and recombined into pLVX-TetOne-Pure vector for myc-tagged Anxa2 over-expression with lentivirus. The primers used within were shown in Table 1. Dual luciferase reporter assay were performed with pNF-κB-TA-luc (Beyotime, Shanghai, China) and pRL-CMV (Promega, WI, USA).

Primers for vector construction.