Immobilon chemiluminescent detection reagent

Cells were lysed directly in M-PER Mammalian Protein Extraction Reagent (Pierce, Waltham, MA, USA) supplemented with Halt Protease and Phosphatase Inhibitors (Pierce, Waltham, MA, USA). For protein extraction from RCC xenograft tissues, specimens were washed with PBS, minced and homogenized using TissueLyser LT (Qiagen, Hilden, Germany) in T-PER Mammalian Protein Extraction Reagent (Pierce, Waltham, MA, USA) containing Halt Protease and Phosphatase Inhibitors (Pierce, Waltham, MA, USA). Quantitation of proteins was made using the bicinchoninic acid (BCA) method (Pierce, Waltham, MA, USA). Proteins were electrophoresed by SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were incubated with the indicated primary antibodies (Cell Signaling Technology Inc, Danvers, MA, USA) and horse-radish peroxidase conjugated secondary antibodies. All proteins were visualised with Immobilon chemiluminescent detection reagent (Millipore, Billerica, MA, USA).

To ensure preservation of protein phosphorylation and high total protein yields, cells were lysed directly in M-PER Mammalian Protein Extraction Reagent (Pierce, Waltham, MA, USA) supplemented with Halt Protease and Phosphatase Inhibitors (Pierce, Waltham, MA, USA). When nuclear and cytoplasmic protein fractions of cells were required, NE-PER Nuclear & Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA) was used. Quantitation of proteins was made using the bicinchoninic acid (BCA) method (Pierce, Waltham, MA, USA). Proteins were electrophoresed by SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were incubated with the indicated primary antibodies (Cell Signaling Technology Inc, Danvers, MA, USA) and horse-radish peroxidase conjugated secondary antibodies. All proteins were visualised with Immobilon chemiluminescent detection reagent (Millipore, Billerica, MA, USA).