FIGURE 2:

Effects of Mib1 overexpression on SMN protein levels and gem number. (A) HEK-293T cells were transfected with either 1 or 2 μg Mib1-myc cDNA. At 24 h after transfection, cells were treated with either vehicle (water) or 1 μM bortezomib and harvested 6 h later. Cell lysates were resolved by SDS–PAGE, and densitometry of the resulting bands was performed using ImageJ software (National Institutes of Health). SMN protein levels were corrected based on actin values. The data represent mean ± SEM of three independent experiments. *, p < 0.05. (B) Overexpression of Mib1 decreases gem number and intensity in cultured cells. HeLa cells were transfected with Mib1-myc or Mib-C1009S-myc for 24 h. The cells were fixed and stained with antibodies to SMN and myc and the appropriate Alexa Fluor secondary antibodies for visualization by microscopy. The number and intensity of nuclear gems were quantitated using Nikon NIS Elements software. At least 300 cells were counted for each transfection condition. That data represent mean ± SEM of three independent experiments. ***, p < 0.001.