Fig. 5

DRAM1 localizes in plasma membrane and regulates the activation of IGF-1 receptors. a Surface of HEK293T cells was biotinylated. The protein levels of EGFR, FLAG and β-actin in whole cell lysate (WCL) and Biotin fractions were detected with immunoblotting. The data showed two independent experiments (1st and 2nd). b HEK293T cells were transfected with FLAG-DRAM1 plasmid for 24 h, the cells were fixed and stained with FLAG antibody (red). Representative immunofluorescent and phase contrast pictures were shown. Arrow indicated the FLAG staining on the membrane. Scale bar represents 10 μm. c and d HEK293T cells were transfected with FLAG empty vector or FLAG-DRAM1 for 36 h (c) or starved for another 18 h (d) before stimulated with IGF-1 (5 ng/ml) for the indicated time. The protein levels of p-IGF-1R β (T1135/36/1150/51), IGF-1R, FLAG and β-actin were detected by immunoblotting. e and f HEK293T cells were transfected with negative control or DRAM1 siRNA for 48 h (e) or stimulated with IGF-1 (5 ng/ml) for the indicated time (f). g HEK293T cells were transfected with negative control or DRAM1 siRNA for 48 h and starved for another 18 h (g) before stimulated with IGF-1 (5 ng/ml) for the indicated time. The protein levels of p-IGF-1R β (T1135/36/1150/51), IGF-1R and β-actin were detected with immunoblotting