Anti p 4ebp1t32 46

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)].

First, cells were washed twice with PBS (Sangon Biotech), lysed in RIPA Lysis Buffer (Beyotime) supplemented with protease inhibitor cocktails and PhosStop (Roche). Protein was mixed with loading buffer, boiled and subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane (Millipore) according to a standard protocol. After blocking with 5% non-fat milk in TBST (50 mM Tris-HCl (Sigma-Aldrich) at pH 8.0, 150 mM NaCl (SinoPharm), 0.1% Tween 20 (Sigma-Aldrich)), the membrane was incubated with non-fat milk or BSA-TBST-diluted primary and secondary antibodies. Signals were detected with ECL detection reagent (Vazyme) using AI600 (GE). The antibodies used in this study were: anti-GAPDH (Proteintech, 10494-1-AP), anti-Pim-1 (D8D7Y) Rabbit mAb (Cell Signaling, 54523 S), anti-c-Myc antibody [Y69] (Abcam, ab32072), anti-c-Myc (phospho S62) antibody (Abcam, ab51156), anti-4E-BP1 (53H11) (Cell signaling, 9644 T), anti-p-4E-BP1(T32/46) (Cell signaling, 2855 T), Anti-rabit IgG, HRP-linked Antibody (Beyotime, A0208).