Rabbit anti lmo1

VLX600 (S8943), DRB18 (S9636), BEZ-235 (S1009) and chloroquine (S6999) were obtained from Selleck Chemicals (Houston, TX, USA). MYCN-amplified neuroblastoma cell lines IMR-32 (Cat. #CCL-127), Sk-N-BE(2) (Cat. #CRL-2271), CHP-212 (Cat. #CRL-2273) and MYCN-nonamplified cell lines SH-SY5Y (Cat. #CRL-2266), Sk-N-AS (Cat. #CRL-2137) were obtained from American Type Culture Collection (ATCC). Primary antibodies used for immunoblotting were the following: mouse anti-MYCN (1:1000, abcam, Cambridge, UK, Cat. #ab16898), rabbit anti-LMO1 (1:1000, Invitrogen, Waltham, MA, USA, Cat. #PA5-77246), rabbit anti-4EBP1 (1:1000, Cell Signaling, Denvers, MA, USA, Cat. #9452), rabbit anti-phospho-4EBP1 (1:1000, Cell Signaling, Cat. #2855), mouse anti-actin (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat. #sc-47778). LC3A/B (1:1000, Cell Signaling Cat. #12741) and MT-COXIV (1:1000, Cell Signaling, Cat. #4850).

Cells were collected and lysed in ice-cold RIPA buffer (with proteinase and phosphor inhibitor) and run on NuPAGE™ 4 to 12% Bis-Tris Mini Protein Gels (Invitrogen) and transferred to nitrocellulose membranes using the iBlot^® 7-Minute Blotting System (Invitrogen). Membranes were pre-treated with SuperSignal^® Western Blot Enhancer Antigen Pretreatment Solution (Thermo Scientific, Waltham, MA, USA) for 10 min, washed according to the manufacturer’s protocol, and subsequently blocked with 5% BSA in 1X PBST for 1 h. The membranes were probed and incubated overnight using the following antibodies in 5% BSA in 1X PBST: mouse anti-MYCN (1:1000, abcam, Cat. #ab16898), rabbit anti-LMO1 (1:1000, Invitrogen, Cat. #PA5-77246), rabbit anti-4EBP1 (1:1000, Cell Signaling, Cat. #9452), rabbit anti-phospho-4EBP1 (1:1000, Cell Signaling, Cat. #2855), mouse anti-actin (1:5000, Santa Cruz Biotechnology, Cat. #sc-47778), LC3A/B (1:1000, Cell Signaling Cat. #12741) and MT-COXIV (1:1000, Cell Signaling, Cat. #4850). The next day, membranes were washed with 1xPBST buffer and incubated with anti-rabbit or anti-mouse secondary antibodies at a 1:5000 dilution for 1 h. SuperSignal^® West Femto Maximum Sensitivity Substrate (Cat. #9452, Thermo Scientific) was used to detect the signal using Amersham Imager 680 (GE Healthcare, Chicago, IL, USA).