Anti dbc1

Protein extracts and immunoprecipitation were resuspended in Laemmli 2X. SDS-PAGE and subsequent protein transfer to PVDF membrane, blocking and washes were performed at standard conditions. Primary antibodies utilized were anti-DBC1 (Bethyl, #A300-434 and #A300-432), anti-SIRT1 (Cell Signalling, #9475), anti-Cyclin D1 (Abcam, #ab137875), anti-β actin (Merck, #A5441), anti-α tubulin (Merck, #T6074), anti-GAPDH (Abcam, #ab9485 and Cell Signalling, #2118), anti-cyclin A2 (Abcam, #ab181591), anti-Ras (Cell Signalling, #3339) and anti-FLAG (Sigma, #F3165). Secondary antibodies utilized were HRP-conjugated IgG from rabbit, mouse and goat (Merck, #A0545, #A9044 and #A8919, respectively). Blot developing was performed with SuperSignal West Pico Chemiluminescent Kit (Pierce, #34080) and results were processed by densitometry analysis with ImageJ (Rasband, W.S., Bethesda, Maryland, USA).

Samples were homogenized in lysis buffer (50‐mM tris, pH 7.5, 150‐mM NaCl, 0.5% NP‐40) supplemented withprotease inhibitor cocktail (MedChemExpress [MCE], HY‐K0010), followed by SDS‐PAGE and electrotransferred to 0.45‐μm polyvinylidene difluoride (PVDF) membrane (Millipore). Membranes were blocked with 5% non‐fat dry milk in tris‐buffered saline with Tween 20 (TBST) for 1 h and then incubated with primary antibodies overnight at 4°C, followed by incubation with proper secondary antibodies. Antibodies were used at the following concentrations: anti‐DBC1 (1:2000, Bethyl, A300434A), anti‐myogenin (MyoG) (1:500, Santa Cruz, sc‐52903), anti‐myosin heavy chain (MHC) (1:1000, Developmental Studies Hybridoma Bank [DSHB], MF20‐C), anti‐FOXO3 (1:2000, Cell Signaling Technology [CST], 2497S), anti‐Atrogin1 (1:500, Santa Cruz, sc‐16806), anti‐Murf1 (1:500, Santa Cruz, sc‐398608), anti‐LC3 (1:500, ABclonal, A19665), anti‐MDM2 (1:500, Santa Cruz, sc‐13161), anti‐ubiquitin (1:500, Santa Cruz, sc‐8017), anti‐β‐tubulin (1:5000, CMCTAG, AT0003) and anti‐GAPDH (1:5000, Millipore, mab374). Blotting signals were visualized on Azure 200 Gel Imager (Azure Biosystems).