Characterization of MDA-MB-231 cells stably expressing GFP-Protrudin. (A) MDA-MB-231 cells stably expressing GFP-Protrudin siRNA resistant to oligo #1 (sires#1) was generated by lentiviral transduction. MDA-MB-231 and MDA-MB-231-GFP-Protrudin sires#1 cells were transfected with two different siRNAs targeting Protrudin (#1 and #2) or control siRNA for 4 d and subjected for Western blotting to detect protein levels of Protrudin, GFP-Protrudin, and actin (loading control) using anti-Protrudin and anti-actin antibodies, respectively. (B) Cells transfected as in A were seeded on coverslips, immunostained for LAMP1, and analyzed by confocal microscopy. Note that LAMP1-positive LE/Lys cluster perinuclearly in Protrudin-knockdown cells as expected, but not in Protrudin/GFP-Protrudin–positive cells, verifying the functionality of GFP-Protrudin.