Effects of FAM188B knockdown on cell EGFR levels and survival signaling pathways. (A,B,D) Cells were transiently transfected with either si-NC or si-FAM188B for 48 h, followed by either attached or suspended culture for 24 h. Cells were then processed for immunoblot analysis using indicated antibodies. The levels of proteins were quantified by densitometry and normalized to GAPDH or Vinculin (Figure S3B–E) (A,D). qRT-PCR assay for relative EGFR mRNA expression levels (B). GAPDH was used as a loading control. (C) Cells were transiently transfected with either si-NC or si-FAM188B for 48 h and then treated with 20 μM MG132 for 2 h before harvest. Cell lysates were processed for immunoblot analysis using indicated antibodies (left panel), and the levels of EGFR were quantified by densitometry and normalized to GAPDH (right panel). Error bars represent standard deviations of the mean of three measurements (* p < 0.05, ** p < 0.01). These experiments were performed three times independently with similar results.