Rabbit polyclonal anti p70 s6 kinase

Cells were lysed with SDS sample buffer (70 mM Tris-HCl, 3% SDS, and 10% glycerol) with inhibitor cocktails of protease and phosphatase. Primary antibodies were rabbit monoclonal anti-phospho-p44/42 kinase (Thr202/Tyr204: Cell Signaling Technology, Danvers, MA, USA), anti-p44/42 kinase (Cell Signaling Technology), rabbit monoclonal anti-phospho-Akt (Ser473:Cell Signaling Technology), rabbit monoclonal anti-Akt (Cell Signaling Technology), rabbit polyclonal anti-phospho-p70 S6 kinase (Thr389: Cell Signaling Technology), rabbit polyclonal anti-p70 S6 kinase (Cell Signaling Technology), and mouse monoclonal anti-β-actin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA). For stimulation experiments, cells were starved for 24 h with serum-free DMEM and then stimulated with 100 ng/ml of CCL5. Cell lysates were obtained after the indicated times stimulation and subjected to SDS-polyacrylamide gel electrophoresis, immunoblotted with the primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and then analyzed by a lumino image analyzer.

To evaluate protein expression on Western blot membranes, the following antibodies were used: rabbit polyclonal anti-LC3 (1:1000) (Novus Biologicals), mouse monoclonal anti-p62 (1:500) (BD 610833), rabbit polyclonal anti-BiP (1:1000) (Cell Signaling), mouse monoclonal anti-CHOP (1:1000) (Cell Signaling), rabbit polyclonal anti-PARP (1:500) (Cell Signaling, 9542), rabbit monoclonal Phospho-Akt (Ser473) (1:500) (Cell Signaling #4060), rabbit monoclonal Akt (1:500) (Cell Signaling #9272), rabbit polyclonal anti Phospho-p70 S6 Kinase (Thr389) (1:500) (Cell Signaling #9234), rabbit polyclonal anti p70 S6 Kinase (1:500) (Cell Signaling #9202), rabbit monoclonal Phospho-4E-BP1 (Thr37/46) (1:500) (Cell Signaling #2855), mouse Monoclonal 4EBP1 (1:500) (Proteintech 60246-1-Ig), rabbit monoclonal Phospho-eIF2α (Ser51) (1:500) (Cell Signaling #3398), rabbit Monoclonal eIF2α (1:500) (Cell Signaling #5324), mouse monoclonal anti-β-actin (1:10000) (Sigma Aldrich) was used as loading control. The goat anti-mouse IgG-HRP (1:30000) (Bethyl Laboratories, A90-116P) and goat anti-rabbit IgG-HRP (1:30000) (Bethyl Laboratories, A120-101P) were used as secondary antibodies. All the primary and secondary antibodies were diluted in 1X PBS–0.1% Tween 20 solution containing 3% of BSA (Serva).