Crki 2

The total protein extraction and western blot were performed, as previously described^{17 (link)}. The primary antibodies were ANXA5 (1:3000, Proteintech, China), p-ERK1/2 (Thr202/Tyr204) (1:1000, Cell Signaling Technology, USA), p-MEK (Ser217 /221) (1:1000, Cell Signaling Technology, USA), DOCK180 (1:400, Proteintech, China), RAC1 (1:800, Proteintech, China), C-MYC (1:800, Proteintech, China), α-Tubulin (1:5000, Proteintech, China), CRKI/II (1:1000, GeneTex, USA). Protein bands were visualized by ECL (Adavansta, USA) and imaged by Bio-Rad ChemiDoc^TM MP system (Bio-Rad, USA).

IHC assay was done to determine the expression changes of ANXA5 (Proteintech, China), RAC1 (Proteintech, China) and CRKI/II (GeneTex,USA) in HCC tissue array, and of CD34 and VEGF-3 (SantaCruz, USA) in primary tissues from Hca-P-ANXA5-shRNA1 and Hca-P-ANXA5-shControl-transplanted mice. Tissue sections were treated with biotin-streptavidin HRP detection systems (ZSGB-BIO, China) according to the manufacturer’s protocol. The images were visualized with 3,3′-diamino-benzidine (DAB) kit (ZSGB-BIO, China) under an BX3-CBH microscope (Olympus, Japan).
IHC immunoreaction intensity was rated into four grades, 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) as Score I. Moreover, based on the detected positively staining cells, DAB staining quantity of each sample was graded as 0 (none), 1 (1–10% cells per field), 2 (10–50%), 3 (51–75%) and 4 (>76%) as Score II. The multiplication of Score I by Score II ranged 0 to 12 was used for IHC immunoreactivity degree. The scores of 0–2, 3–5, 6–8, and 9–12 were considered as negative (−), weak (+), moderate (++) and strong (+++). IHC assay was scored by two independent experienced pathologists, separately.