Total phospho mtor

Cells were washed twice with cold PBS, and lysed in RIPA lysis buffer containing protease and phosphatase inhibitors to extract total protein. Cell lysates were centrifuged for 5 min (12,000 g, 4°C), and the supernatant was collected. Protein concentrations were determined by Bio-Rad protein Assay kit (Bio-Rad, Philadelphia, PA, United States). Equal amounts of protein (50 μg) were separated on a 10% SDS–PAGE gel, and transferred to a nitrocellulose (NC) membrane at 300 mA and 4°C for 1 h. The membrane was incubated with primary antibody (1:1000), and then with a fluorescence-conjugated secondary antibody (1:10000). The primary antibody against PRMT5 was purchased from Merck Millipore Ltd., (Germany); antibodies against H4R3me2s and H4 were purchased from Abcam (Cambridge, MA, United States); antibodies against FGFR3 and eIF4E were purchased from Santa Cruz Biotechnology (Dallas, TX, United States); antibodies against total/phospho-AKT, total/phospho-ERK and total/phospho-mTOR were purchased from Cell Signaling Technology (Danvers, MA, United States). GAPDH was used as the loading control and for normalization. The signal intensity of the membranes was detected with a LI-COR Odyssey Scanner (Belfast, ME, United States).

Proteins were extracted from A549 cells using RIPA lysis buffer containing protease and phosphatase inhibitors. After two washes with cold PBS, the cell lysate (12,000× g, 4 °C) was centrifuged for 5 min, and the supernatant fraction was collected. The protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Philadelphia, PA, USA). The extracted proteins were separated in 10% SDS-PAGE gels (50 μg) and transferred to nitrocellulose (NC) membranes at 300 mA for 1 h at 4 °C. The proteins were then incubated with a primary antibody (1:1000) and a fluorescence-coupled secondary antibody (1:10,000). Additionally, eIF4E antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies against total/phospho-AKT, total/phospho-ERK, and total/phospho-mTOR were obtained from Cell Signaling Technology (Danvers, MA, USA).