Anti gapdh antibody

30 µg of total proteins extracted from AtT-20 cells and primary cultured cells were separated on SDS/polyacrylamide gels and transferred to a nitrocellulose filter. USP8 and POMC antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA) and diluted at 1:200; FLAG antibody was from Merck KGaA (Darmstadt, Germany) and diluted 1:1000; SSTR5 antibody was from Abcam (Cambridge, UK), cyclin E and cyclin D3 antibodies were from Cell Signaling Technology (Danvers, MA, USA) and were diluted 1:1000. Primary antibodies were incubated o/n at 4 °C. Anti-GAPDH antibody (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) was used at 1:4000 for 1 h at room temperature. Secondary antibodies, anti-mouse or anti-rabbit (Cell Signaling Technology, Danvers, MA, USA) were incubated at room temperature for 1 h at 1:2000. Chemiluminescence was detected with ChemiDOC-IT Imaging System (UVP, Upland, CA, USA). Densitometrical analysis was performed with NIH ImageJ software. All of the original Western Blot figures shown in Figure S1, Figure S2, Figure S3, Figure S4, Figure S5 and Figure S6.

Anti-FLAG antibody (Sigma-Aldrich, A8592), anti-GFP antibody (Roche, 11,814,460,001), anti-GAPDH antibody (Ambion, AM4300), anti-H4 (Millipore, 07-108) and oligos (Sigma-Aldrich) were used.

Cells were incubated 10 min at 37°C with forskolin 1 μM (Sigma-Aldrich, St. Louis, MO, USA) or BIM53097 100 nM (kindly provided by Biomeasure Incorporated/IPSEN, Milford, MA, USA), while to perform time course experiments related times are indicated in the corresponding figures. For cyclin D3 and p27 expression levels analysis cells were treated with BIM53097 100 nM for 3 h and 48 h, respectively.
Total proteins extracted from cultured cells were quantified by BCA assay, separated on SDS/polyacrylamide gels and transferred to a nitrocellulose filter. Phospho-Filamin A (Ser2152), Filamin A, phospho-ERK1/2, ERK1/2, c-Myc and cyclin D3 antibodies (Cell Signaling Technology, Danvers, MA, USA) were used at 1:1,000. P27 and D2DR antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) were diluted at 1:200. Phospho-AKT antibody was diluted at 1:2,000 and AKT at 1:1,000 (Immunological Science, Rome, IT). Primary antibodies were incubated overnight at 4°C, secondary antibodies anti-rabbit or anti-mouse (Cell Signaling Technology, Danvers, MA, USA) were used at 1:2,000 at room temperature for 1 h. Anti-GAPDH antibody (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) was used at 1:4,000 for 1 h at room temperature. Chemiluminescence was detected trough a ChemiDOC-IT Imaging System (UVP, Upland, CA, USA) and then analysed with NIH ImageJ software.

Cells or homogenized mouse tissues were lysed in radio-immunoprecipitation assay buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing Halt Protease Inhibitor (ThermoScientific). Protein lysates (5 µg) were separated by 4–12% Bis-Tris SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Invitrogen). Membranes were probed with 1∶1000 rabbit anti-mouse SAMHD1 antiserum, 1∶40,000 anti-GAPDH antibody (Ambion) and 1∶1000 anti-human SAMHD1 antibody (Origene) followed by 1∶4,000 horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin serum and 1∶40,000 HRP-conjugated goat anti-mouse immunoglobulin serum (Sigma). Membranes were developed with chemiluminescent substrate (Pierce) exposed to Hyperfilm (GE Healthcare). Band intensities were quantified using ImageJ software [44] .