P ulk1s757

Total cell lysates were prepared and analyzed by western blotting as described previously [32 (link)]. The proteins were recovered in sodium dodecyl sulfate (SDS) buffer, separated by SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. The membrane was blotted with appropriate primary and secondary antibodies. Rabbit polyclonal antibodies against ULK1, p-ULK1^S757, FLT3, p-FLT3, STAT5, p-STAT5, p-MEK, p-ERK, caspase-9, caspase-3, PARP, AMPKα, p-AMPKα^T172, mTOR, p-mTOR^S2448, p70S6K, p-p70S6K^T389, PERK, and p-eIF2a were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies against LC3 and p-ATG13^S318 were obtained from Novus Biologicals (Littleton, CO, USA). Mouse anti-p62/SQSTM antibodies were procured from Abnova (Taipei, Taiwan). Anti-p-PERK^T982 antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti-p-ULK1^S555 and mouse anti-α-tubulin monoclonal antibodies were obtained from Merck Millipore (Billerica, MA, USA). The secondary antibodies were coupled to horseradish peroxidase. The blots were visualized using an enhanced chemiluminescence (GE Healthcare Bio-Sciences; RPN2232).

Paraffin-embedded tissue sections were prepared as described previously (Guo et al., 2013 (link)) for H and E, and IHC staining. Antibodies utilized for IHC were Atg7 (Sigma Aldrich, A2856, RRID:AB_1078239), Lkb1 (Santa Cruz Biotechnology, sc-32245, RRID:AB_627890), p62 (Enzo Life Sciences, PW9860-0100, RRID:AB_2877676), p-AMPK^Th172 (Cell Signaling, 2535S, RRID:AB_331250), p-ACC^S79 (Cell Signaling, 3661, RRID:AB_330337), p-S6^S235/236 (Cell Signaling, 4858, RRID:AB_916156), p-ULK1^S555 (Cell Signaling, 5869, RRID:AB_10707365), p-ULK1^S757 (Cell Signaling, 14202, RRID:AB_2665508), LC3 (Nano Tools, LC3-5F10, RRID:AB_2722733), Ki67 (Abcam, ab-15580, RRID:AB_443209), cleaved caspase-3 (Cell Signaling, 9661S), OLFM4 (Cell Signaling, 39141, RRID:AB_2650511), lysozyme (Agilent, A0099, RRID:AB_2341230), and p53 (Novus Biologicals, NB200-103SS, RRID:AB_2877680). Paraffin embedded tissue sections were used for the TUNEL assay by means of the HRP-DAB TUNEL staining kit (ab206386) and the slides were counterstained by methyl blue following the protocol provided by the TUNEL staining kit. For the quantification of IHC and TUNEL assay, tissues were analyzed by quantifying at least 10 images at 20x magnification. A minimum of 200 cells were scored for each image.

Western blotting was performed in whole cell lysates of AsPC1, BxPC3, L3.6pl, MIAPaCa2, and PO2 cells treated with 2.5, 5, and 7.5 µM PVT for 24 h SDS-PAGE resolved proteins were immunoblotted for ULK1, pULK1(S757), Atg101, FIP200, Beclin-1, Atg-5, LC3A/B, cleaved caspase-3 and β-actin (Cell Signaling Technologies, Danvers, MA, USA). All experiments were repeated for at least 2 times.

Hippocampal proteins and cellular proteins were extracted in RIPA lysis buffer containing 1 mM PMSF (Beyotime, China). The proteins were separated by 8–12% SDS-PAGE and electrotransferred onto PVDF membranes. After being blocked with 5% skim milk, the membranes were incubated with primary antibodies against DOR, LC3 (1:1000, Abcam), P62, β-actin, AMPKα, p-AMPKα T172, mTOR, p-mTOR S2448, ULK1, p-ULK1 S317, and p-ULK1 S757 (1:1000, Cell Signaling Technology, USA) overnight at 4 °C. Afterwards, they were incubated with secondary antibodies for 2 h at room temperature and then scanned with a Bio-Rad gel imaging system.

Antibodies against mTOR (1:1000), p-mTOR (Ser2448) (1:1000), p-ULK1 (S757) (1:1000), Akt (1:1000), p-Akt (S473) (1:1000), NF-κB(p65) (1:1000), p-p65 (S536) (1:1000), acetylated lysine (Ace-lys) (1:1000), and Beclin1(1:1000) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Antibodies targeting Col II (1:1000 for western blotting analysis, 1:200 for immunohistochemistry staining), ULK1 (1:1000), and p62 (1:1000) were purchased from Abcam Inc. (Cambridge, MA, USA). Antibodies against Aggrecan (1:1000 for western blotting analysis, 1:200 for immunohistochemistry staining) and β-actin (1:40,000) were purchased from Sigma-Aldrich in China (Shanghai, China). Rabbit IgG and Protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against LC3B (1:1000 for western blotting analysis, 1:200 for immunohistochemistry staining), Lamin B1 (1:1000), and β-tubulin (1:1000) were purchased from Novus Biologicals, Inc. (Littleton, CO, USA), Protein Tech Group (Chicago, IL, USA), and Sangon Biotech (Shanghai, China), respectively. Other reagents were of the highest grade commercially available.