Chemidoc xsr

^TAMRA−ubiquitinated PCNA was prepared as described34 (link) and mixed to USP4 CD and FL at the described concentrations. Aliquots of the reaction were loaded on SDS–polyacrylamide gel electrophoresis after being stopped with loading buffer at different time points. Bands corresponding to ^TAMRA−ubiquitinated PCNA (S) and ^TAMRA−ubiquitin (P) were quantified using ChemiDoc XSR (Bio-Rad). Product concentration was calculated as S₀ · (P/(P+S)), where S₀ is the initial substrate concentration.

Western blotting was performed according to manufacturer’s instructions (Bio-Rad laboratories, Hercules, CA, USA). In brief, 20–40 μg of proteins was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Criterion TGX anykD acrylamide separating gel Bio-Rad). The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA) according to the manufacturer’s instructions. The membranes were dried and then incubated for 1h using primary antibodies for sirtuin proteins (SIRT1, SIRT2, SIRT3, SIRT4, SIRT6), Santa Cruz Biotechnology, Santa Cruz, CA, USA and glucokinase (GCK) Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBS (20 mM TRIS, pH = 7.6, 137 mM NaCl), 0.04% Tween-20 and 5% low fat milk and then incubated for 30 min with secondary antibodies conjugated with horseradish peroxidase (anti-rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBS, immunoblots were visualized by means of a chemiluminescence reaction (Millipore) by Image Lab^TM Software (Bio-Rad) under a luminescent image analyzer (Chemidoc XSR + Bio-Rad, Philadelphia, PA 19103, USA). Only bands below the saturation limit were analyzed and shown. β-actin (Sigma-Aldrich, S.r.l., Milan, Italy) was used as loading control.

Protein expression analysis was performed according to manufacturer’s instructions (Bio-Rad). In brief, proteins were subjected to SDS-PAGE (4–20% acrylamide separating gel, Criterion stain-free TGX Biorad). The separated proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA), which was incubated overnight with the diluted antibody. Primary antibodies against CREB, pCREB, pERK, Sirt1, and c-Fos, and secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA); CAMKII, pCAMKII, ERK, and PKC were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Immunoblots were visualized by means of a chemiluminescence reaction (Millipore) by Image LabTM Software (Biorad) under a luminescent image analyzer (Chemidoc XSR+ Bio-Rad). Only bands below the saturation limit were analyzed. The protein level was normalized to the optical density of total proteins in each lane.