Figure 3.

GLS and ELS share the same topology of ER/Golgi localization as mTOR. (A and B) Subcellular fractionation of GLS-EGFP and ELS-EGFP. GLS-EGFP and ELS-EGFP were transiently expressed in HEK293T cells. Lysates of these cells were fractionated by differential centrifugation. T, total cell lysates; P10, 10,000g pellet; S10, 10,000 × g supernatant; P100, 100,000 × g pellet; S100, 100,000 × g supernatant. (C and D) GLS-EGFP and ELS-EGFP are localized on the cytoplasmic face of Golgi and ER membranes. The P100 pellets from A and B were treated with different concentrations of proteinase K (0, 5, and 50 μg/ml) in the presence or absence of the detergent Triton X-100 and analyzed by Western blot with antibodies specific for GFP.