Anti p mtor ser2448

The following antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA): goat anti-COL1A1, anti-p-p70s6k (Thr389), and anti-Akt; mouse anti-α-SMA, anti-LC-3, anti-mTOR, and anti-GNMT; rat anti-CD3; and rabbit anti-p70s6k, anti-p62, anti-p-mTOR (Ser2448), and anti-COL4A2. Mouse anti-p-Akt (Ser473) and rabbit ant-iNOS and anti-MPO antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Rat anti-F4/80 and rabbit anti-ICAM-1 and anti-VCAM-1 antibodies were obtained from Abcam (Cambridge, MA, USA). Both the mouse anti-GAPDH antibody and Masson’s trichrome staining kit were obtained from Sigma–Aldrich (St. Louis, MO, USA). The mouse anti-TGF-β antibody was obtained from R&D Systems (Minneapolis, MN, USA).

The specimens were lysed in a lysis buffer, and quantification of the protein content in the homogenate was performed using a BioRad protein assay kit. In the experimental procedure, 30 μg of the protein were applied to 10% SDS-PAGE gels and subjected to electrophoresis, and after protein separation, PVDF membranes (Millipore, Billerica, MA, USA) were utilized for protein transfer via immunoblotting. The antibodies utilized in this study were anti-PDK1 Ser 241 (3061), anti-p-P70S6 Thr389 (9205), anti-p-P6 Ser235/236 (3061), anti-p-4EBP1T32/46 (2855), anti-p-elf4e (9741) procured from Cell Signaling and anti-14-3-3 (Sc-1657), anti-p-FKHR Ser 256 (SC-101681), anti-p-NFκB (SC-271908), anti-Nrf2 (SC-722), anti-HO1 (SC-136961), anti-p-AKT Ser 473 (SC-SC-798), anti-p-mTOR Ser 2448 (SC-293132), and anti-β-Actin from Santa Cruz Biotechnology, California, USA, all employed at a dilution of 1:1000. Subsequently, the membranes were subjected to incubation with secondary antibodies and the protein bands were observed through the employment of an ECL detection reagent. The X-ray films were subjected to scanning, and the optical densities of the bands were quantified relative to the corresponding β-actin band through the utilization of the ImageJ software, as previously explicated [55 (link)].

After deparaffinization of the sections, antigen retrieval was performed by microwaving in 10 mmol/L citrate buffer (pH 6.0) for p-Akt, p-S6 and LC3 staining, and by heating in Tris-ethylenediaminetetraacetic acid buffer (pH 9.0) with a pressure cooker for p-mTOR and Ki-67 staining. After blocking the endogenous peroxidase, the sections were incubated overnight at 4°C with anti-p-mTOR (Ser2448) (1∶20; Santa Cruz Biotechnology, Inc.), anti-p-Akt (Ser473) (1∶25), anti-p-S6 (1∶200), anti-LC3 (1∶100; Nanotools) and anti-Ki-67 (pre-diluted) antibodies. Then the sections were incubated with the secondary antibody conjugated to the peroxidase-labeled polymer, EnVison+system (DakoCytomation). Color development was performed using DAB, and the sections were counterstained with hematoxylin. Control sections were evaluated by substitution of the primary antibodies with nonimmunized serum without signal detection.