Anti vps34

Anti-p62, anti-caspase-3, anti-Beclin-1, anti-Vps34, and anti-Atg5 antibodies were purchased from Cell signaling. Anti-LC3 antibody and necrostatin-1 were purchased from Sigma. Doxorubicin, chloroquine diphosphate (CQ), doxycycline, and cycloheximide were purchased from Calbiochem. zVAD was purchased from R&D system. 20(S)-ginsenoside Rg₃ was isolated by prep-LC. Glutathione S-transferase (GST)-TRAIL was obtained as described previously [67 (link)].

The cells were washed twice with PBS containing 1 mmol/l phenylmethylsulfonylfluoride (PMSF). Next, the cells were scraped off the dishes and pelleted at 500×g for 10 min. The cells were subsequently lysed in cold lysis buffer (20 mmol/l Tris-HCl, 1 mmol/l EDTA, 150 mmol/l NaCl, 1 mmol/l EGTA, 1% Triton X-100, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l Na3VO4, 1 µg/ml leupeptin and 1 mmol/l PMSF) and sonicated for 5 s. The lysates were clarified by centrifugation at 12000×g for 30 min at 4°C. Equal amounts of cell lysate were resolved by 8 or 15% SDS-PAGE. The membranes were blocked for 1 h in 5% powdered milk in TBST (10 mmol/l Tris-HCl, 150 mmol/l NaCl and 1% Tween-20). The membranes were subsequently immunoblotted using anti-Ambra1, anti-Beclin1, anti-LC3, anti-PARP, anti-Cathepsin D, anti-ATG5, anti-VPS34 and anti-β-actin antibodies (Cell Signaling Technology, Danvers, MA, USA). The PVDF membranes were incubated with a horseradish peroxide-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Immunoreactive bands were exposed to film (Kodak, Rochester, NY, USA). The band intensity was semi-quantified using BandScan software after scanning the blots (V300; EPSON, Tokyo, Japan).

Anti-Vps34 (1:1,000, no. 4263), Beclin 1 (1:1,000, no. 3738), acetylated lysine (1:500, no. 9441), haemagglutinin (HA)-Tag (1:1,000, no. 3724, Rabbit) and HA-Tag (1:1,000, no. 2367, Mouse) antibodies were purchased from Cell Signaling Technology. Anti-UVRAG (1:1,000, NBP1–18885), Beclin 1 (1:1,000, NBP1–00088) and LC3 (1:5,000, NB100–2220) antibodies were obtained from NOVUS. Anti-Rubicon (1:1,000, ab92388) and p150 (1:1,000, ab128903) antibodies were obtained from Abcam. Anti-SIRT1 (1:1,000, AJ1717a), SIRT2 (1:1,000, AJ1718a) and HDAC1 (1:1,000, AP1101a) antibodies were purchased from Abgent. Anti-p300 (1:500, sc-585), p62/SQSTM1 (1:2,000, sc-28359) and HDAC2 (1:1,000, sc-55542) antibodies were purchased from Santa Cruz Biotechnology. Anti-a-tubulin (1:2,000, T6199), Flag (1:5,000, F3165) and Atg14L (1:1,000, A6358) antibodies were obtained from Sigma. Anti-phospho S409 Beclin 1 antibodies were generated by immunizing rabbits with the corresponding phosphopeptides. Bafilomycin A1, rapamycin, deacetylase inhibitors TSA and NAM, and CK1 inhibitor D4476 were purchased from Sigma.

Recombinant human IL-6 was from Pepro Tech. LC3B; 3-MA and baf A1 were from Sigma Aldrich. Anti-ERK1/2, anti-phospho-ERK1/2, anti-Akt, and anti-phospho-Akt antibodies were from Bioworld Technology. Anti-STAT3, anti-phospho-mTOR, anti-mTOR, anti-p62, anti-Bcl-2, anti-Beclin 1, and anti-VPS34 antibodies were from Cell Signaling Technology (CST). Anti-phospho-STAT3 was from Wuhan Boster Biological Technology, Ltd. The STAT3 inhibitor LLL12 was from Merck Millipore. pcDNA3.1(+) (V790-20) was from Invitrogen.

Anti-LC3B, anti-VPS34, anti-JNK, pJNK, AKT, pAKT antibodies were obtained from Cell Signaling Technology, Massachusetts, USA; secondary antibodies labeled with horseradish peroxidase were purchased from Promega, Madison, WI. The apoptosis inhibitor ZVAD-FMK was purchased from Merk, Darmstadt, Germany. BafilomycinA1 was purchased from InvivoGen, San Diego, CA, USA.