Azure 600 western blot imaging system

A total of 10 μg of protein extracts were separated by 10% SDS-PAGE Tris-glycine polyacrylamide gel electrophoresis, and 0.2 μm nitrocellulose membranes were used for the transfer in Towbin buffer for 4 h at constant 280 mA. Blots were incubated for 5 min with Ponceau S (0.1% (w/v) Ponceau S in 5% glacial acetic acid) for total protein visualization to control for possible loading differences. For immunodetection of biotinylated proteins, membranes were blocked in 7% milk in 1xTBS and 0.01% Tween-20 and streptavidin-HRP immunostaining (1:5,000, Invitrogen cat. #19534–050) was performed at room temperature for 1 h in blocking solution. After 3 washes with TBST, membranes were covered with SuperSignal West Femto Maximum Sensitive Substrate (Thermo Scientific, Cat. #34095) according to manufacturer’s instructions and chemiluminescence was then documented using Azure 600 Western Blot Imaging System (Azure Biosystems).

Patient-derived tumor cells for immunoblotting were lysed in Triton X-100 lysis buffer containing protease inhibitors and sonicated. Collected protein lysates were stored at −20 °C. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Cat #23227; Thermo Fisher Scientific). An amount of 15 μg of total protein was size fractioned by 12.5% SDS-PAGE. Electrophoresis-separated proteins were electrically transferred to a polyvinylidene difluoride (PVDF) membrane, washed in PBST buffer, and blocked in 2% fat-free milk for 1 h at room temperature, then incubated with primary antibodies at 4 °C overnight. Following primary antibody blotting, specific signal was detected with species-appropriate peroxidase-conjugated secondary antibody (Thermo Fisher Scientific) using SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat #34580; Thermo Fisher Scientific) and imaged using an Azure 600 Western blot imaging system (Azure Biosystems, Dublin, CA, USA). Details regarding antibodies used for Western blots can be found in Table S3.

Cells were lysed by Western Blot Cell Lysate Buffer according to the manufacture’s instruction. Protein concentrations of cell lysates were determined by using BCA Protein Assay Kit. After denaturing proteins by heat-inactivation in loading buffer, each sample was loaded on SDS polyacrylamide gels and separated by electrophoresis. Thereafter, proteins were transferred to PVDF membranes by using Bio-Rad Trans-Blot Turbo system. Subsequently, membranes were blocked with Tris-buffered saline buffer containing 5% non-fat milk and 0.1% Tween-20 at 4 °C overnight. Then membranes were incubated with primary antibodies (CREB1 #9197, cell signaling, and anti-LaminB1 #ab194109, Abcam) and anti-rabbit peroxidase-conjugated secondary antibody (#7074, cell signaling) stepwise. Between any steps, membranes were washed by TBS–Tween buffer extensively. Finally, the signal was detected with ECL WB Detection Kit by Azure600 Western Blot Imaging System (Azure Biosystems).