Radioimmunoprecipitation assay buffer

A-549 cells transfected with the empty vector pcDNA3.1, pcDNA3.1-DLC1, pcDNA3.1-DLC2 or pcDNA3.1-DLC3 were lysed using radioimmunoprecipitation assay buffer (CWBiotech) for protein extraction. Total protein was quantified using a bicinchoninic acid assay and 20 µg protein/lane was separated by a 10% SDS-PAGE. The separated proteins were subsequently transferred onto a PVDF membrane and blocked in 5% non-fat milk at 25°C for 1 h. The membranes were incubated with primary antibodies against DLC1 (cat. no. ab126257; 1:500; Abcam), DLC2 (cat. no. ab126489; 1:500; Abcam), DLC3 (cat. no. 13899-1-AP; 1:400; ProteinTech Group, Inc.) and GAPDH (cat. no. 10494-1-AP; 1:5,000; ProteinTech Group, Inc.) overnight at 4°C. Following the primary incubation, membranes were incubated with horseradish peroxidase-labeled secondary antibodies (cat. no. A00001-2; 1:5,000; ProteinTech Group, Inc.) for 1 h at room temperature. Protein bands were visualized using the enhanced chemiluminescence system. Protein expression was density-quantified using ImageJ software (version 1.49; National Institutes of Health) with GAPDH as the loading control.

Total cell protein was extracted with radioimmunoprecipitation assay buffer (CWBIO, Beijing, China) and the protein concentration was detected by the BCA kit (CWBIO). Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. These membranes were blocked with 5% skimmed milk powder for 1 hour, washed several times with Tris-buffered saline and Tween 20, then incubated with primary antibodies against TXNIP (dilution 1:1000; Cell Signaling Technology Inc., Danvers, MA, USA) and β-actin (dilution 1:1000; Cell Signaling Technology Inc.) at 4°C overnight. They were then incubated with secondary antibodies conjugated to horseradish peroxidase (dilution 1:5000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 2 hours, and protein bands were visualized with ECL reagent (GE Healthcare, Chicago, IL, USA) and imaged by the typhoon scanner (GE Healthcare). β-actin was used as an internal reference.