Phospho p70 s6 kinase t389

For immunoblotting, the following antibodies were used: a mouse monoclonal antibody against heat shock protein 90 kDa (610419; BD Transduction Lab) and rabbit polyclonal antibodies against PFAS (A17517; Abclonal), 4E-binding protein 1 (9452; Cell Signaling Technology), p70 S6 Kinase (9202; Cell Signaling Technology), phospho-p70 S6 Kinase (T389) (9234; Cell Signaling Technology), LC3 (63 (link)), TSC2 (3990; Cell Signaling Technology), CAD (11933; Cell Signaling Technology), and DHODH (14877-1-AP; Proteintech). For secondary antibodies, horseradish peroxidase–conjugated anti-mouse (111-035-003; Jackson ImmunoResearch Laboratories, Inc) and anti-rabbit (111-035-144; Jackson ImmunoResearch Laboratories, Inc) IgGs were used.

LPA-induced signaling was further examined by determining the ratio of the phosphorylated form of each protein to the total amount of the protein using Western analysis. Whole cell lysates were separated by 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane using a transfer apparatus according to manufacturer’s protocol (Bio-Rad, Hercules CA). After incubation with 5% bovine serum albumin (BSA, fraction V; Sigma) in PBST (10 mM sodium phosphate, pH 8, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was rinsed once with PBST and incubated with antibodies against phosphorylated CREB (S133) (1:1000, #9191), phospho-Erk1/2 (Erk1/2; Thr202/Tyr204; 1:1000, #9101) and Erk1/2 (1:1,000, #9102), phospho-p70S6kinase (T389) (1:1000, #9205 and #9234), p70 S6 kinase (T389) (1:1000, #9205 and #9234) (all from Cell Signaling Technology, Danvers MA), phospho-p70 S6 kinase (1:1000, MAB8963, R&D Systems), GAPDH (1:20000, sc47724, Santa Cruz Biotechnology, Dallas, TX) at 4 °C overnight. Membranes were washed three times for 10 min and incubated with 1:5000 dilution of anti-rabbit or anti-mouse secondary antibodies for 1 h. The membrane was washed three times for 15 min with PBS and detected using a chemiluminscent detection system (ECL; Thermo Fisher) using manufacturer’s protocol.

10–20 mg of protein per lane was separated by standard SDS–PAGE and transferred onto PVDF membranes. After blocking in blocking solution [100 mM Tris/HCl (pH 8.0), 450 mM NaCl, 5% dry milk and 0.05% Tween-20], the membranes were incubated with antibodies against: LC3B (1:2500), phospho-T389 p70 S6 kinase (1:2500), p70 S6 kinase (1:2500), phospho-S437 AKT (1:2500), FOXO3A (1:2500), FOXO1 (1:2500) (all from Cell Signaling), AKT (1:1000), SREBP-1 (1:1000) (both from Santa Cruz) and SREBP2 (1:1000; R&D Systems). Equal loading of protein was verified by the detection of β-Actin (1:10,000; Sigma Aldrich). Peroxidase-conjugated anti-mouse (1:5000), anti-rabbit (1:5000; both from KPL Sera Care) secondary antibodies were used for detection of specific signals with Pierce ECL Western Blotting Substrate (Thermo Scientific) (Supplementary Fig. S10).

Preparation of whole cell lysates and immunoblotting was performed as previously described in [18 (link)]. Antibodies used: ATR (1:1000; 13934S), phospho-S428 ATR (1:2500; 2853S), phospho-S1981 ATM (1:2500; 4526S), ATF-4 (1:1000; 11815S) cleaved caspase-3 (1:1000; 9661S), PARP1 (1:1000; 9542S), phospho-S345 CHK1 (1:1000; 2341S), CHK1 (1:1000; 2345S), CHK2 (1:1000; 2662S), CHOP (1:1000; 2895S), BIM (1:2500; 2933S), LC3B (1:1000; 2775S), phospho-S15 p53 (1:2500; 9284S), phospho-S9 GSK3β (1:2500; 9323S), phospho-S473 AKT (1:2500; 4060S), AKT (1:1000; 2920S), phospho-T389 p70 S6 kinase (1:2500; 9205S), p70 S6 kinase (1:2500; 9202S) (all from Cell Signaling), GRP78 (BiP; 1:1000; AF4846; R&D systems), ATM (1:2500; NB100-309, Novus Biologicals), p53 (1:5000; sc-965), BRCA1 (1:500; sc-6954), DR5 (1:1000; sc-166624), LAMP1 (1:2500; sc-20011) (all from Santa Cruz), p62 (1:2500; PM045, MBL) and phospho-S139 (γH2AX; 1:2500; 07–164, Merck Millipore). Equal loading of protein was verified by the detection of vinculin (1:5000; sc25336, Santa Cruz), β-actin (1:10,000; A5441) and α-tubulin (1:10,000; T5168) (both from Sigma Aldrich). Images were quantified using ImageJ 1.8 (NIH).