Anti smc1

Nuclear Cytosolic Fractionation Kit (BioVision) was used to obtain protein extracts. Protein concentration was determined using the Bio‐Rad DC Protein Assay (Bio‐Rad). Up to twenty micrograms of nuclear protein extracts was separated in SDS–polyacrylamide gels by electrophoresis. After protein transfer onto nitrocellulose membrane, the membranes were incubated with the indicated antibodies. Antibody binding was detected after incubation with a secondary antibody coupled to horseradish peroxidase using chemiluminescence with ECL detection KIT (GE Healthcare).
Primary antibodies: anti‐TRF1 1:1,000 (BED5, Bio‐Rad), anti‐TRF1 1:500 (homemade), anti‐SMC‐1 1:2,000 (Bethyl), anti‐AKT1 1:500 (Millipore), anti‐p‐AKT 1:500 (Ser473, Cell Signaling Cell Signaling Technology), anti‐S6 1:500 (Cell Signaling Cell Signaling Technology), anti‐p‐S6 1:500 (Ser240/244, Cell Signaling Technology), anti‐TIN2 1:1,000 (Abcam), anti‐RAP1 1:1,000 (Bethyl).
For the quantification, protein‐band intensities have been quantified by densitometric analysis with ImageJ software. The Trf1 total levels have been normalized versus SMC1 and the mean of the Trf1/SMC1 ratio deriving from at least 3 different replicates has been used to generate the chart.

Lymphocytes were lysed in F buffer (1×10⁷ cells mL⁻¹) and subjected to immunoblot analysis as described previously [3] , [10] . Total ACC and ACC^S79 were detected by bi-fluorometric analysis using the Odyssey LICOR system and ImageJ software was used for integral signal quantification. Anti-AMPKα1 and anti-ACC^S79 were kindly provided by Grahame Hardie, University of Dundee, U.K. Anti-Smc1 was purchased from Bethyl Laboratories Inc and anti-Hif1α was obtained from R&D Systems. Anti-Glut1 was a gift from Geoff Holman, University of Bath, U.K. [19] , [24] (link). All other antibodies for immunoblotting were obtained from Cell Signaling Technology. Antigens were detected using suitable HRP-conjugated secondary antibodies and enhanced chemoluminescence.