Ab102005

TRIzol reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The PrimeScript One Step miRNA cDNA Synthesis kit and the SYBR premix Ex Taq™ II qPCR reagents were purchased from Takara Bio, Inc. (Otsu, Japan). An rno-miR-383 mimic kit (cat. no., miR1003114-1-2) and a corresponding negative control (cat no., miR01201-1-2) were procured from Guangzhou Ruibo Biological Technology Co., Ltd. (Guangzhou, China). A Transwell chamber migration assay kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). A Boyden chamber invasion assay kit was purchased from EMD Millipore (Billerica, MA, USA). β-actin and primary antibodies for western blot against β-actin (ab8227), APRIL (ab64967), MCL-1 (ab28147) and COX-2 (ab102005) were obtained from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated-Goat Anti-Rabbit immunoglobulin (Ig)G (cat. no. ab6721) used as a secondary antibody was obtained from Abcam.

Protein lysates were used to extract the proteins in the tumor tissues. Protein concentrations were determined by the bicinchoninic acid (BCA) colorimetric protein assay method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to resolve the proteins, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Merck, Darmstadt, Germany) at 80 V for 30 min. Membranes were blocked with 5% nonfat dried milk powder in Tris-buffered saline (TBS) and Tween 20 (TBST) solution for 1 hr. The primary antibodies included a rabbit polyclonal anti-COX-2 antibody (1: 1000) (ab102005, Abcam) an anti-Ang1 antibody (1: 1000) (Ab183701, Abcam), an anti-Ang2 antibody (1: 500) (ab8452, Abcam), an anti-HIF-1α antibody (1: 1000) (ab82832, Abcam), and anti-VEGF antibody (1: 2000) (ab39256, Abcam), which were incubated on the membranes overnight at 4°C. The membranes were incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 2000) (Proteintech, Chicago, Ill, USA) for 1 h. The enhanced chemiluminescence (ECL) system was used to detect the signals on the membranes. The protein expression level was standardized with GAPDH, and quantification of band intensity was performed by Image J software (National Institutes of Health, USA).