Anti p70 s6k

Medium was removed, and cells were carefully washed with PBS before the addition of cold N-PER buffer (ThermoFisher) containing a cocktail of protease and phosphatase inhibitors (Cell Signaling #5872). Cell lysates were clarified by centrifugation at 4 °C and stored in aliquots at − 80 °C. NuPAGE 4–12% Bis-Tris gels (ThermoFisher) were loaded with 2 μg of sample protein per lane and ran at constant voltage for 1.5–2 h before blotting to either nitrocellulose or PVDF membrane using the iBLOT2 system (ThermoFisher). The antibodies used were anti-phospho-PDK1 (pSer241); anti-phospho-SGK (pSer422); anti-mTOR; anti-mTOR pSer2481 (SAB4504514, SAB4503834, SAB4501038, SAB4301526 Sigma-Aldrich); PDK1 (MA5–15797, ThermoFisher); SGK1; b-actin; anti-phospho-S6 ribosomal protein (Ser235/236) (#12103, #8457, #2211 cell signaling); and anti-p70 S6K (#05-781R, Millipore). Band intensities were quantified using ImageJ version 1.51 software (Schneider et al., 2012 (link)).

The plumbous acetate [Pb(Ac)₂] for Pb²⁺ supply was purchased from Sigma–Aldrich. Dulbecco's Modified Essential Medium (DMEM) and FBS were purchased from GIBCO Invitrogen. The Lyso-Tracker Red probes for acidic lysosome staining were from Beyotime. Anti-GRP78 (glucose-regulated protein 78), anti-GRP94, phosphorylates eukaryotic initiation factor 2 (anti-peIF2a), anti-cleaved caspase-3, anti-light chain 3 (anti-LC3) and anti-pS6 were purchased from Cell Signaling Technology. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase), BCL2-associated X protein (anti-Bax), B-cell lymphoma 2 (anti-BcL2) and anti-p70S6K (S6 kinase 1) antibodies were from Millipore. The p62 antibody was from Santa Cruz. Other reagents were of the highest purity available.