Gfp tag

SDS-PAGE and western blotting was carried out using standard methods. The following antibodies were used: PIM1 (12H8, Santa Cruz), PIM1 (A300–313A, Bethyl Laboratories), Actin (ab6276, Abcam), GAPDH (G8795, Sigma), SUMO2 (51–9100, Zymed), His-tag (27-4710-01, GE Healthcare), HA-tag (12CA5, Sigma), Flag-tag (F1804, Sigma), MYC-tag (9E10, Hybridoma supernatant), GFP-tag (sc-8334, Santa Cruz), GST-tag (sc-459, Santa Cruz), total ERK1/2 (ER16, Transduction lab), and phospho S10 Histone H3 (06–570, Millipore). The following antibodies were purchased from Cell Signaling Technology: phospho S62 c-MYC (13748), total c-MYC (5605), total Histone H3 (4499), phospho S112 Bad (5284), total Bad (9239), phospho T37/46 4E-BP1 (2855), total 4E-BP1 (9644), phospho T389 p70S6K (108D2), total p70S6K (49D7), phospho S473 AKT (D9E), total AKT (40D4), phospho T202/Y204 ERK1/2 (9106) and phospho tyrosine-100 (9411). Sheep polyclonal UBC9 and chicken polyclonal RNF4 were from Ron Hay, University of Dundee. Secondary antibodies were purchased from Biorad and Thermo Fisher Scientific.

The cells were washed for three times by sterile and cold PBS, then the cells were lysed by Western and IP cell lysis buffer (Beyotime, 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100). The concentrations of the proteins were measured through BCA assay (Pierce). The same amount of protein samples were mixed with 2× SDS loading buffer, and they were loaded into SDS-PAGE. Through Bio-Rad Trans Blot Turbo System, the proteins were transferred to PVDF membrane (Millipore, Burlington, MA, USA) by the semidry process. The membranes were blocked in 5% skimmed milk solution at room temperature for 90 min and incubated with suitable primary antibodies at 4°C overnight. The primary antibodies used in this study were against Myc, Flag, HA, GFP Tag (Santa Cruz, Santa Cruz, CA, USA), GAPDH, and tubulin. Using the TBST buffer, the membranes were washed for three times, and they were then incubated with the secondary antibody at room temperature on the rocker platform for 60 min. Finally, proteins were detected by WesternBright™ ECL (Advansta, San Jose, CA, USA), and cold CCD camera was used for digital imaging.

Co-immunoprecipitation was performed to determine the direct or indirect interaction of ORFV002 and S100A4 in eukaryotic forms. HEK293T cells were co-transfected with either p002EGFP, pEGFP-N1 and pCMV-Tag2B-S100A4, or either pCMV-Tag2B-S100A4, pCMV-Tag2B and p002EGFP. The cells were washed twice with cool PBS and lysed with 1 ml cool lysis buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF) for 10 min on ice at 24 h post-transfection, followed by centrifugation at 14000 × g for 10 min. Three micrograms of antibodies against GFP-tag (Santa Cruz Biotechnology; USA) or FLAG-tag (ABclonal; USA), or isotype IgG incubated with 30 μl pre-treated Beaver Beads Protein A/G (Beaver Nano Technology, China) for 2 h at 4°C. The obtained supernatant was immunoprecipitated with the washed Beads-antibody overnight at 4°C. After washing and centrifugation, the pellet was examined by western blot using anti-GFP or anti-FLAG antibodies.