Beclin1 nb500 249

Cells were homogenised in lysis buffer (RIPA buffer; Sigma-Aldrich), Ethylenediaminetetraacetic acid free protease inhibitor (Roche Pharmaceuticals), Phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich) and protein levels quantified by bicinchoninic acid assay (Thermo Fisher Scientific). For western blot analysis samples were probed overnight with primary antibody: Human SIRT1 (#9475; 1:1000; Cell Signalling Technology, MA, U.S.A), Mouse SIRT1 (07–131; 1:1000; Millipore, Massachusetts, U.S.A) or Beta-actin (β-actin) (A2228; 1:20000; Sigma-Aldrich), COL2A1 (SAB4500366; 1:1000; Sigma-Aldrich), SOX-9 (ab26414; 1:1000; Abcam, Cambridge, U.K.), ULK1 (NBP2-24738; 1:1000; Novus Biologicals, Cambridge, UK), Beclin1 (NB500-249; 1:1000; Novus Biologicals), LC3 (NB100-2220; 1:1000; Novus Biologicals), ATG5 (#12994; 1:1000; Cell Signalling Technology); ATG7 (#8558; 1:1000; Cell Signalling Technology), Acetyl-Lysine Antibody (#9441; 1:1000; Cell Signalling Technology), P62/SQSTM1 (NBP1-48320; 1:1000; Novus Biologicals). Secondary horseradish peroxidase (HRP)-conjugated anti-rabbit (#7074; 1:5000; Cell Signalling Technology), Secondary HRP-conjugated anti-mouse (#7076; 1:5000; Cell Signalling Technology)

SNAP25 (GTX113839), NSE (GTX101553), LDH (GTX101416), NBR1 (GTX114539) and HO-1 (GTX101147) from GeneTex, Inc.(Nottingham, UK); PSD95 (clone 6G6-1C9, MA1-045) from ThermoFisher Scientific (Waltham, MA, USA); GAPDH (G9545) and LC3B (L7543) from Sigma-Aldrich; p62 (ab56416), Nrf2 (ab31163), NQO1 (ab34173), CathD (ab6313) and TATA-binding protein TBP (ab51841) from Abcam (Cambridge, UK); Mfn2 (NBP1-96547), ATG9A (NB110-56893), Ambra1 (NBP1-07124) and Beclin1 (NB500-249) from Novus Biologicals (Abingdon, UK); Keap1 (10503-2-AP) from Proteintech (Manchester, UK); COXIV (4844) and Parkin (4211) from Cell Signalling; p62-S403 (D343-3) and p62-S351 (PM074) from Medical and Biological Laboratories Co., Ltd (Nagoya, Japan); K48-linkage-specific polyubiquitin antibody (05-1308) from Millipore; OPTN (H-220) (sc99214) from Santa Cruz (Heidelberg, Germany).

Glyoxal solution (GO), MGO solution, and d-(+)-Glucose were purchased from Sigma (St. Louis, MO, USA). Bovine serum albumin (RD tech, C0082—100), Matrigel matrix (Invitrogen, MA, USA), Endothelial cell growth medium 2, and MV 2 (PromoCell, Heidelberg, Germany) were also purchased from respective companies. p38 (9212S), phospho-p38 (p-p38; 9211S), ERK1/2 (9102S), phospho-ERK1/2 (p-ERK1/2; 9101S), JNK (9252S), phospho-JNK (p-JNK; 9251S), mTOR (7C10, 2983s), phospho-mTOR (p-mTOR, Ser2448, 2971s), Akt (9272S), phospho-Akt (p-Akt; 9271S), Caspase-3 (9661S), and Alexa Fluor® 555 Phalloidin (8953S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Bcl-2 (sc-492), Bax (sc-493), and VEGF-C (sc-9047) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Beclin-1 (NB500249) was purchased from Novus Biologicals (Littleton, CO, USA). Hoechst 33342 (Waltham, MA, USA) was purchased from Thermo Fisher Scientific. Cyto-ID® autophagy detection kits were obtained from Enzo Life Science (Farmingdale, NY, USA). p62 (610833) and fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit I (San Diego, CA, USA) were purchased from BD Biosciences Pharmingen.