Figure 4(A) Mouse embryonic fibroblast (MEF) cells were transfected with HA-tagged ArfGAP1. Immunofluorescence staining was assessed for HA-tagged ArfGAP1 (blue), mTOR (green), and LAMP2 (red, lysosomal marker). Left- A representative example of the immunofluorescence staining (scale bar, 10 μm). The top left image shows the staining of HA-tagged ArfGAP1, with a white outline highlighting the region that is positive for HA-tagged ArfGAP1. This outline was overlaid with the mTOR channel (top right) and the LAMP2 channel (bottom left). The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm (Rizk et al., 2014; bottom right). The co-localization of mTOR and LAMP2 was analyzed for the region within the outline (HA-ArfGAP1 positive) and the region outside of the outline (HA-ArfGAP1-negative), respectively. Right- In each microscopy field, the image was processed as described above, and the co-localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm (Rizk et al., 2014). The quantification was performed for the subgroups of cells that are positively or negatively stained for HA-tagged ArfGAP1, respectively, and compared using Student's t-test. Columns represent Mean ± SEM (N = 3). (B) Wildtype (WT) human embryonic kidney 293A (HEK293A) cells were transfected of HA-tagged ArfGAP1, or HA-tagged ArfGAP2 as a control. Cells were cultured under normal conditions (NC), or starved of amino acids (-AA) for 2 hours, and then stimulated with amino acids for 1 hour. Immunofluorescence analysis was performed for HA-tagged ArfGAP1, HA-tagged ArfGAP2, mTOR, and LAMP2. In each microscopy field, the colocalization between mTOR and LAMP2 (normalized by the LAMP2 positive puncta) were quantified for HA-tagged ArfGAP1 or HA-tagged ArfGAP2 positive or negative cell populations, respectively. Bar plot shows Mean ± SEM (N = 8~10). P values were determined using Student's t-test, and ns denotes for not significant. (C) HEK293A cells were transfected with HA or HA-tagged Raptor. Cells were cultured under normal condition (NC) or starved of (-AA) for 2 hours. Endogenous ArfGAP1 was immunoprecipitated (IP). IP or whole cell lysate (WCL) samples were probed for HA and ArfGAP1. The phosphorylated of S6K1 at threonine 389, S6K1, and Actin served as a loading control. (D) MIA Paca-2 cells stably expressing FLAG-tagged or HA-tagged TMEM192 cells were cultured under normal condition (NC) or starved of amino acids (-AA), and then stimulated with 2 mM glutamine and asparagine (+Gln/Asn) for 2 hours. HA-tagged TMEM192 was immunoprecipitated (IP) with HA beads and IP and whole cell lysates (WCL) were probed for ArfGAP1, HA, RagA, or p18 (LAMTOR1). Flag, pS6K1 (mTORC1 activity), S6K1, and Vinculin were probed for as controls. (E) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1 or EGFP (control) with HA-tagged Raptor or HA-tagged Raptor fused with C-terminus region of Rheb (HA-Raptor-C-Rheb), and Myc-tagged S6K. Cells were starved of amino acids (-AA) for 2 hours and then 4 mM of glutamine (+Gln) or asparagine (+Asn) were added for 2 hours. Cell lysate were immunoprecipitated (IP) with anti-Myc beads. IP or whole cell lysate (WCL) samples were probed for HA, Myc, and phosphorylated S6K1 at threonine 389 (mTORC1 activity). Actin served as a loading control.