rabbit anti phospho mtor  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: miR-378a and NPNT coordinate autophagy regulation in podocytes through mTOR and MAPK signaling

doi: 10.64898/2026.03.19.709781

Figure Lengend Snippet: a. Autophagic flux assay by western blot analysis. Cultured human podocytes were transfected with miR-378a mimic or miR-Ctrl and treated with 30 nM bafilomycin A1 (BafA) for 46 h to block autophagosome-lysosome fusion or left untreated. LC3-II protein accumulation was assessed by Western blot. Autophagic flux was calculated by subtracting basal LC3-II levels from LC3-II levels after BafA treatment within each group. Protein expression was normalized to GAPDH. Autophagy flux is given as fold change compared to miR-Ctr, n = 4 independent experiments b. Loss-of-function analysis. Inhibition of endogenous miR-378a using a specific antisense inhibitor decreased autophagic flux in podocytes, confirming a positive regulatory role for miR-378a in autophagy. n =4 independent experiments, c. Western blot analysis of mTOR phosphorylation at Ser2448 in podocytes following miR-378a mimic or miR-Ctrl transfection. Phospho-mTOR/total mTOR ratio was normalized to Vinculin and given as fold change compared to miR-Ctrl. n = 3 independent experiments, ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies used in this study: Phospho-Akt (Ser473, #9271S, Cell Signaling), AKT (#9272, Cell Singaling), phospho-EGFR (Tyr1068, #3777S, Cell Signaling), EGFR (#2232, Cell Signaling), phospho-ERK1/2 (phosphor-p44/42 MAPK (Thr202/Tyr204), #9106, Cell Signaling), ERK1/2 (p44/42 MAPK, #9102, Cell Signaling), GAPDH (sc-32233, Santa Cruz), LC3 (NB100-2220, Novus), phospho-mTOR (Ser2448, #5536S, Cell Signaling), mTOR (2983S, Cell Signaling), vinculin (V9131, Sigma-Aldrich).

Techniques: Flux Assay, Western Blot, Cell Culture, Transfection, Blocking Assay, Expressing, Inhibition, Phospho-proteomics

Journal: bioRxiv

Article Title: miR-378a and NPNT coordinate autophagy regulation in podocytes through mTOR and MAPK signaling

doi: 10.64898/2026.03.19.709781

Figure Lengend Snippet: a. Autophagic flux analysis of cultured human podocytes after transfection with NPNT-specific siRNA (NPNT siRNA) or non-targeting control siRNA (Ctrl siRNA) +/- 30 nM BafA for 44 h to block autophagosome-lysosome fusion. LC3-II protein accumulation was assessed by Western blot. Autophagic flux was calculated by subtracting basal LC3-II levels from LC3-II levels after BafA treatment within each group. Protein expression was normalized to GAPDH. Autophagy flux is given as fold change compared to Ctr siRNA, n = 4 independent experiments. b. Cells as in a. were stimulated with NPNT protein or unstimulated +/- 30 µM BafA Autophagic flux was assessed as in a., n = 5 independent experiments. c. Western blot analysis of mTOR phosphorylation at Ser2448 in podocytes following NPNT siRNA or Ctrl siRNA transfection. Phospho-mTOR/total mTOR ratio was normalized to Vinculin and given as fold change compared to miR-Ctrl. n = 3 independent experiments. ** p < 0.01 *** p < 0.01

Article Snippet: Primary antibodies used in this study: Phospho-Akt (Ser473, #9271S, Cell Signaling), AKT (#9272, Cell Singaling), phospho-EGFR (Tyr1068, #3777S, Cell Signaling), EGFR (#2232, Cell Signaling), phospho-ERK1/2 (phosphor-p44/42 MAPK (Thr202/Tyr204), #9106, Cell Signaling), ERK1/2 (p44/42 MAPK, #9102, Cell Signaling), GAPDH (sc-32233, Santa Cruz), LC3 (NB100-2220, Novus), phospho-mTOR (Ser2448, #5536S, Cell Signaling), mTOR (2983S, Cell Signaling), vinculin (V9131, Sigma-Aldrich).

Techniques: Cell Culture, Transfection, Control, Blocking Assay, Western Blot, Expressing, Phospho-proteomics