Anti srpk1

Cells with stable overexpression or knockdown of USP39 were collected by RIPA buffer. Immunoprecipitation was conducted with anti-HA (Abcam) or anti-SRPK1 (Santa Cruz) or anti-Pan-phospho-SR (Santa Cruz). By incubation with protein A agarose (Santa Cruz), the antibodies were removed. Proteins were prepared and separated by 10% SDS-PAGE. The interaction between USP39 and SRPK1/SRSF1 was analyzed by Western blot using anti-Flag tag (Abcam) or anti-SRSF1 (Abcam).

Cells with stable overexpression or knockdown of USP39 were lysed using RIPA lysis buffer (Beyotime, China), and the protein concentration was measured by BCA assay (Beyotime). Samples were prepared in SDS sample loading buffer, and transferred to the PVDF membrane. The main antibodies of Western blot were anti-USP39 (Abcam), anti-SRSF1 (Abcam), anti-SRPK1 (Santa Cruz), anti-VEGFA_165b (R&D, MAB3045), and anti-VEGF-A (Abcam, ab214424). The membrane was blocked by 5% milk (nonfat) in TBS (0.05% Tween 20) at room temperature for 1 h, and incubated with the primary antibodies for 2 h and with HRP-conjugated IgG (rabbit). Western blotting detection system (Tanon) was applied to detect Chemiluminescence of the membrane.

GES-1, an immortalized human gastric epithelial cell line, was also purchased from ATCC, and cells (passages 5–10) were maintained in Gibco RPMI-1640 (Thermo Fisher Scientific, Inc.) medium supplemented with 10% FBS, 2 mM L-glutamine (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin. The human gastric cancer cell lineMGC803, BGC823and HGC27 were obtained from American Type Culture Collection (ATCC, Manassas, Va.), and was cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 100 U/mL penicillin/streptomycin (Gibco) and were maintained in a humidified atmosphere with 5% CO₂ at 37°C. All experiments were performed in the absence of FBS. Recombinant human IGF-1 was obtained from Sigma Corporation (St. Louis, USA). All antibodies in this experiment can be purchased from Santa Cruz Biotechnology (Santa Cruz, USA), involving anti-p-AKT, anti-t-AKT, anti-SRPK1 (Santa Cruz Biotechnology Cat. number sc-289; 1:200); anti-N-cadherin,anti-MMP2,anti-Slug, and anti-β-actin. All assays were repeated three times.