A0760 40

Primary antibodies: rabbit‐anti‐MAD2 (1:2,500; ab70385, abcam); mouse‐anti‐αTubulin DM1A (1:2,000, T9026‐100UL, Sigma‐Aldrich); mouse‐anti‐HSP70 (1:3,000; ab47455, abcam); rabbit‐anti‐GFP (1:5,000; ab290, abcam); rabbit‐anti‐BubR1 (1:1,500; A300‐386A, Bethyl Laboratories Inc.); mouse‐anti‐βActin (1:2,000; A0760‐40, USBiological Life Sciences); mouse‐anti‐Adaptin β (1:500; 610382, BDbiosciences); mouse‐anti‐GAPDH (1:2,000; sc‐47724, SantaCruz Biotechnology); rabbit‐anti‐p31^comet (human) (1:1,000; provided by Dr. Hongtao Yu; UT Southwestern, Dallas, Texas, USA); and rabbit‐anti‐CNX (1:2,000; ab22595, abcam). Secondary antibodies: HRP‐linked anti‐mouse IgG (1:5,000; 7076S, Cell Signaling); HRP‐linked anti‐rabbit IgG (1:5,000; 7074S, Cell Signaling); and IRDye 800CW goat‐anti‐Rabbit IgG (1:5,000; 925‐32211, Li‐Cor).

Proteins were purified from 3t3 cells and BLA tissue. Cells and tissue were lysed using a lysis buffer containing Tris pH 7.4 50 mM, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, protease inhibitors cocktail (Roche, Mannheim, Germany) and protease and phosphatase inhibitors cocktail (ThermoScientific, Rockford, USA)). Cell/tissue homogenate was sonicated and total protein content was quantified using a commercial kit (Pierce^TM BCA Protein Assay Kit, Thermoscientific) with assistance of the software GEN5 (BioTek, Vermont, USA).
Western Blot analysis was conducted using the following primary antibodies and dilutions: anti-PTEN: 1:1000, ab154812 (Abcam, Cambridge, UK), anti-beta actin 1:2000, A0760–40 (USBiological, Massachusetts, USA) and anti-GAPDH 1:3000, ab9485 (Abcam), anti-CREB 1:250, 48H2 (Cell Signalling, Boston, Massachussets). Secondary antibody signals were detected using the Clarity^TM Western ECL detection kit (BioRad, California, USA) and the software Fluorchem^TM HD2 (Alpha Innotech, Biozym, Vienna, Austria). Quantification of optical densities was performed using the program ImageJ (Maryland, USA) and values were normalized to those of housekeeping genes (beta actin and GAPDH).

A previously described procedure was used24 with the following primary antibody (SERT: 1:1000, sc‐1458, Santa Cruz Biotechnology; Flotillin‐1:1:1000, ab133497, Abcam; β‐actin: 1:2000, A0760‐40, US Biological, Salem, Massachusetts) which were incubated with membranes overnight at 4°C. Target protein densitometry values were normalized to those of the housekeeping protein to semi‐quantitatively determine protein levels.