P mtor s 2448

Cell lysis and Immunoblotting was performed as described previously (8 (link)). 30 μg of protein was loaded per lane in 8–10% SDS gels. The following antibodies were used to probe the membranes: p-ACC (S-79) (#11818 Cell signaling), p-mTOR (S-2448) (#ab109268 Abcam), mTOR (#ab2732 Abcam), p-LKB1 (S-428) (#3482 Cell signaling), LKB1 (#3050 Cell signaling), p-AMPK (T-172) (#2535 Cell signaling), p-AMPK (S-173) (#ab55886 Abcam), GAPDH (#2118 Cell signaling), Vinculin (sc-5573, Santa Cruz Biotechnologies), p-TAK1 (#4508 Cell signaling), TAK1 (#5206 Cell signaling), p-CAMKII (#12716 Cell Signaling). For Figures 1, 5, Western blots were developed using near-IR-labeled secondary antibodies and quantitated on a Li-COR Odyssey CLx and analyzed using ImageStudio v5.2. Other blots were developed on film and analyzed semi-quantitatively. All in vitro experiments were repeated 2–4 times with consistent results observed.

P-PDK1(Ser241), Akt, p-Akt(Ser473), 4EBP1 and p-4EBP1(Ser65) and α-tubulin were purchased fromCell Signaling Technology (Danvers, MA, USA). P70S6K and p-P70S6K(Thr470) were acquired from Millipore (Boston, MA, USA). Antibodies against LC3, SQSTM1, Atg5, Beclin1, LAMP1, Bcl-2, Bax, cytochrome C, active caspase-9, active caspase-3, cleaved PARP, mTOR, p-mTOR(S2448) and β-actin were obtained from Abcam (Cambridge, UK). YZT was dissolved in DMSO and diluted with culture medium. CQ, 3-methyladenine (3-MA) and rapamycin (RAPA) were bought from Sigma-Aldrich (Saint Louis, MO, USA).

The antibodies used for immunocytochemistry as well as for western blot analysis in this study were as follows: ATG7 (8558, Cell Signaling Technologies, Danvers, MA, USA); BAG3 (ab47124, Abcam, Cambridge, UK); BCL2 (sc-492, Santa Cruz, Dallas, TX, USA); ERα (RM9101S0, Thermo Fischer Scientific, Waltham, MA, USA); ERK1/2 (9102, Cell Signaling Technologies); p-ERK1/2 (9106, Cell Signaling Technologies); LC3B (L7543, Sigma-Aldrich); mTOR (OP97, Millipore, Billerica, MA, USA); p-mTOR S2448 (ab51044, Abcam); NBR1 (00004077-M01, Abnova, Taipei, Taiwan); SQSTM1 (GP62-C, Progen, Heidelberg, Germany); PI3K Class III (4263, Cell Signaling Technologies); Tubulin (T9026, Sigma-Aldrich); WIPI1 (HPA007493, Sigma-Aldrich); Beclin1 (ab51031; Abcam); p70S6K (9202, Cell Signaling); p70S6K Thr389 (9206, Cell Signaling). Secondary antibodies were anti-mouse/rabbit/guinea pig antibodies conjugated to DyLight 488, 649 and Cy3 (immunofluorescence, Jackson ImmunoResearch, West Grove, PA, USA) or to HRP (immunoblotting, Jackson ImmunoResearch).