Anti phospho mtor ser2481

Insulin, human recombinant from Saccharomyses cerevisiae, was purchased from Sigma-Aldrich. Doxorubicin was obtained from Tocris. Anti-survivin, anti-caspase-3 (cleaved form), anti-phospho-p53 (Ser¹⁵), anti-phospho-Akt (Ser⁴⁷³), anti-Akt, anti-phospho-mTOR (Ser²⁴⁸¹) and anti-mTOR antibodies were obtained from Cell Signaling. Anti-Sp1, anti-phospho-p70S6K (Thr⁴²¹/Ser⁴²⁴), anti-p70S6K, anti-β-actin, anti-GAPDH, anti-Smac/DIABLO antibodies and p70S6K inhibitor PF4708671 were purchased from Santa Cruz Biotechnology. Anti-VDAC1, anti-p53, anti-Bcl-2, and anti-BAX antibodies were obtained from Abcam. PI3K inhibitor LY294002 and mTOR inhibitor Rapamycin were purchased from Calbiochem. Anti-cytochrome c antibody was included in ApoAlert Cell Fractionation Kit (Clontech).

Human CRP protein was purchased from Millipore. To remove sodium azide from the commercial CRP preparation, CRP was repeatedly filtered with Tris buffer (10 mM Tris, 100 mM NaCl and 2 mM Ca²⁺) until remaining 0.0001% sodium azide using Ultrafilter Vivaspin 500 (Sartorius). Anti-survivin, anti-phospho-p53 (Ser15), anti-p53, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-mTOR (Ser2481) and anti-mTOR antibodies were obtained from Cell Signaling. Anti-PTEN, anti-phospho-p70S6K (Thr421/Ser424), anti-p70s6k, anti-phospho-ERK1/2 (Tyr204), anti-ERK1/2 and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology. BpV (PTEN inhibitor), U0126 (ERK inhibitor), SP600125 (JNK inhibitor), CGK733 (ATM/ATR inhibitor) and NU7026 (DNA-PK inhibitor) were purchased from Calbiochem. PFT-α (p53 inhibitor) was obtained from Sigma-Aldrich.

A Western blot (WB) analysis was performed as described previously [50 (link)]. The level of klotho was assessed using an anti-klotho antibody (Abcam, Cambridge, MA). The levels of p21, p53, IL-1, TGF-β and MCP-1 were assessed using anti-p21, p53, IL-1, TGF-β and MCP-1 antibodies, respectively (Abcam, Cambridge, MA). The levels of LC3 I/II and Beclin1 were assessed using anti-LC3A/B and anti-Beclin1 antibodies, respectively (Cell Signaling, Beverly, MA). The levels of the phosphorylated and total proteins for mTOR were assessed using anti-phospho mTOR Ser2448, anti-phospho mTOR Ser2481 and anti-mTOR antibodies (Cell Signaling, Beverly, MA, USA). The levels of the phosphorylated and total proteins of AMPK and ULK1 were assessed using anti-phospho AMPKα Thr172, anti-AMPKα, anti-phospho ULK1 and anti-ULK1 antibodies (Cell Signaling, Beverly, MA, USA). The level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was assessed using an anti-GAPDH antibody (Bioworld Technology Inc., MN, USA) as a loading control. The blots were visualized using an enhanced chemiluminescence detection system (Tanon-5200Muilti, China). The densitometric analysis was performed using ImageJ Software.