β actin signal

Western blot analyses were performed to study protein expression within the callus tissue. The analyses included the assessment of the expression of stromal cell-derived factor (SDF)-1α and aspartyl aminopeptidase Dnpep, which have both been reported to be regulated by miR-140 [19 (link),23 (link)]. After saving the whole-protein fraction, proteins were separated and transferred to membranes by standard protocols. Proteins were then probed using anti-SDF-1α (sc-74271, 1:50; Santa Cruz Biotechnology, Heidelberg, Germany) and anti-Dnpep (1:50; Abcam, Cambrigde, UK) antibodies. The appropriate peroxidase-conjugated anti-IgG antibodies served as secondary antibodies (1:1500; Dako, Hamburg, Germany and 1:1000; R&D Systems, Wiesbaden, Germany). To visualize protein expression, luminol-enhanced chemiluminescence was performed after exposure of the membrane to the Intas ECL Chemocam Imager (Intas Science Imaging Instrument GmbH, Göttingen, Germany). Detected signals were normalized to β-actin signals (1:5000; Sigma-Aldrich, Taufkirchen, Germany) to correct for unequal loading.

Aliquots of cell lysates, containing 40 μg of proteins, were subjected to SDS polyacrylamide gel electrophoresis and proteins were blotted on a polyvinylidene difluoride (PVDF) membrane using a Trans Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were probed with the following primary antibodies SIRT3, SIRT1, LC3B, Cyclin B1, and p53 (1:1000, Cell Signaling Technology, Danvers, MA, USA); p21/WAF1/Cip1 clone CP74, Cyclin E, and Cyclin D1/D2 (1:1000, EDM Millipore Temecula, CA, USA); Cdk1/2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA); p27 (1:1000, Abcam, Cambridge, MA, USA). After incubation with a correspondingly suited horseradish peroxidase-conjugated secondary antibody (1:2500, Cell Signaling Technology, Danvers, MA, USA), chemiluminescent signals were developed using the Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) and captured with the ChemiDoc imaging system XRS + (Bio-Rad Laboratories, Hercules, CA, USA). To read band intensity of the target proteins, images were analyzed using the Image Lab software (version 6.0, Bio-Rad, Hercules, CA, USA). The intensity of the relevant bands was normalized to the β-actin signal (1:5000, Sigma Aldrich, St. Louis, MO, USA).