Visionworks ls acquisition and analysis software

After treating spheroids with visfatin (100 ng/mL) for 48 h, spheroids were lysed in lysis buffer. Proteins were separated on 4-20% Mini-Protean TGX Precast Protein Gels (Bio-Rad) and then transferred to Trans-Blot Turbo Mini PVDF transfer packs (Bio-Rad) using the Trans-Blot Turbo transfer system (Bio-Rad). The blots were blocked for 1 h with 0.02 M Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 and then incubated overnight at 4°C with antibodies specific for MMP2 (#4022; Cell Signaling Technology), GLUT1 (ab652; Abcam), GLUT4 (#2213; Cell Signaling Technology), hypoxia-inducible factor (HIF-1; #41493; GeneTex), and visfatin (NAMPT; ab233294; Abcam). The membranes were then washed three times in Tris-buffered saline and 0.1% Tween 20 and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit (#7074) or anti-mouse (#7076) secondary antibodies (Cell Signaling Technology). β-actin (A5316; Sigma-Aldrich) was used as a loading control. Immunopositive bands were visualized using WesternBright Sirius HRP substrate (Advansta, Menlo Park, CA, USA). Quantification of protein bands from three independent experiments was performed by densitometry using VisionWorks LS Acquisition and Analysis software (UVP, Upland, CA, USA).