Ab133485

Western blotting was followed, was described elsewhere [12 (link)]. Polyclonal rabbit antibody for Fli-1 (ab133485), PKCδ (ab182126), Phospho- PKCδ (ab133456), MEK (ab178876), Phospho-MEK (ab96379), BAD (ab32445), and Phospho-BAD (ab129192) were obtained from Abcam (Abcam, Cambridge, UK); ERK (#4695) and phospho-ERK (#9101) from Cell Signalling Technology (CST, Danvers, MA01923, USA), β-actin (20536–1-AP) and GAPDH (13937–1-AP), from Proto-Technology (Protein-Tech, Bucuresti, Romania). Antibody dilution according to the manufacturer instructions.
The inhibitor of MEK (U0126) [#S1102] were obtained from Sellectchem (Sellectchem, Houston, USA) and PKC agonist Phorbol 12-myristate 13-acetate (TPA) from Sigma, (Sigma, St. Louis, MO, USA). These compounds were dissolved in DMSO and added to the cells at indicated concentration, as described in the results section.

Total protein from cell lines was extracted using RIPA buffer (Beyotime Institute of Biotechnology, CN) containing 1:100 PMSF (Solarbio, CN). The protein concentration was determined using a BCA kit (Solarbio, CN) according to the manufacturer's protocol. Load equal amounts of protein into the wells of the SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked using non-fat milk for 1 h at room temperature. Incubate the membrane with primary antibody in blocking buffer overnight at 4°C. After washed by TBST (Beyotime Institute of Biotechnology, CN) at room temperature for three times, the membrane was incubated with Anti-rabbit IgG (H + L) DyLight™ 800 4X PEG Conjugated secondary antibody (5151s, Cell Signaling Technology, US) in blocking buffer at room temperature for 1 h. The following primary antibodies were used: anti-FLI1 (ab133485, Abcam, UK), anti-UBASH3A (15,823–1-AP, Proteintech, DE), anti-UBASH3B (19,563–1-AP, Proteintech, DE), polyclonal rabbit test primary antibodies; anti-GAPDH (G9545, Sigma Aldrich, US). Antibody dilution was conducted according to the manufacturer’s instructions. The Odyssey system (LICOR Biosciences) was used for western blot membrane imaging and analysis.

Protein from A673/TR/shEF1 cells was extracted at d0, d7, d11, d14, and d17 with RIPA and anti-protease cocktail (Roche). Western blots were performed following routine protocols and specific band detection was achieved by the use of rabbit monoclonal anti-FLI1 antibody (1:1000, ab133485; Abcam)^{38 (link)}, rabbit polyclonal anti-MYBL2 antibody (1:500, sc-725; Santa Cruz)^{39 (link)}, and mouse monoclonal anti-ß-actin (1:10,000, A-5316; Sigma-Aldrich). Anti-rabbit IgG horseradish peroxidase-coupled antibody (1:3000, Amersham Bioscience) and anti-mouse IgG horseradish peroxidase coupled antibody (1:3,000; Amersham Bioscience) was used as secondary antibody. Proteins were visualized using chemiluminescence (Pierce ECL Western blot chemiluminescent substrate; Thermo Fisher Scientific).