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. Author manuscript; available in PMC: 2016 Aug 11.
Published in final edited form as: Cell Rep. 2016 Jul 28;16(6):1510–1517. doi: 10.1016/j.celrep.2016.07.008

Figure 2. LRS is necessary for amino acid activation of PLD1.

Figure 2

HEK293 cells were transduced with shLRS or control lentiviruses as described in Figure 1 legend. (A) Cells were stimulated with AA as described in Figure 1 legend, followed by in vivo PLD assays. (B) Cells were stimulated with Leu as described in Figure 1 legend. (C) Cells were treated as in (A) except that siRNA for IRS and EPRS were introduced by transfection. (D) Cells were transduced with shLRS lentivirus, and then transfected with HA-PLD1, followed by AA stimulation and immunostaining with anti-HA and anti-LAMP1/2 antibodies. (E) Cells were treated as in (D) without transfection, and immunostained with anti-mTOR and anti-LAMP1/2 antibodies. For D and E, representative images and quantification of co-localization (overlap coefficient) are shown. (F) Cells were transduced with shRNA lentiviruses as indicated, and stimulated with amino acids with or without 100 μM PA. All data shown are mean ± SD or representative blots from 3 to 5 independent experiments. *P<0.05; **P<0.01. Data denoted by different letters (a, b) are significantly different (P ≤ 0.05). Scale bars, 5 μm; (enlarged images) 0.5 μm.