Journal: Scientific reports

Article Title: Neuroglobin regulates autophagy through mTORC1/RAPTOR/ULK-1 pathway in human neuroblastoma cells.

doi: 10.1038/s41598-025-91701-w

Figure Lengend Snippet: Fig. 5. NGB overexpression regulates autophagy targeting mTOR signaling pathway. CTRL and stably transfected SH-SY5Y-NGB-FLAG cells were lysed in lysis buffer. The lysates were analyzed by western blot to detect P-Ser2448-mTOR or total mTOR protein levels, using anti-P-Ser2448-mTOR pAb or anti-total mTOR pAb or P-Ser757-ULK1 or total ULK1 using anti-P- Ser757-ULK1 pAb. Loading control was evaluated using mouse anti-Beta-Actin mAb. Results represent the mean ± SD from three independent experiments (n = 3). *** p < 0.001. Uncropped blot images are shown in Supplementary Information file.

Article Snippet: Membranes were blocked with 5% w/v non-fat dried milk (sc-2325, Santa Cruz Biotechnology, Dallas, Texas, USA) in Tris buffer saline (1706435, Bio-Rad Laboratories) (TBS), containing 0.05% v/v Tween 20 (1706531, Bio-Rad Laboratories) (TBST) and probed with rabbit anti-LC3 pAb (NB100-2331, Novus Biologicals, Centennial, Colorado, USA), rabbit anti-SQSTM1 mAb (Cell Signaling Technology, Danvers, Massachusetts, USA), rabbit anti-P-Ser757-ULK1 pAb (6888, Cell Signaling Technology), rabbit anti-P-Ser2448-mTOR pAb (2971, Cell Signaling Technology), rabbit total ULK1 mAb (8054, Cell Signaling Technology) or rabbit anti-total mTOR mAb (2983, Cell Signaling Technology).

Techniques: Over Expression, Stable Transfection, Transfection, Lysis, Western Blot, Control