Anti s6k

All plasmids used in this study were sequenced to confirm their identity. Crbn, Ampk, Slo, or HA-Ubiquitin constructs used in this study were described previously^{26 (link)}. Orai1 (MMM1013-202764440), Orai2 (MMM1013-202859855), and Orai3 (MMM1013-202842392) cDNA were purchased from Open Biosystems to construct expression vectors by a PCR-based strategy. Glutamine synthetase (GS) cDNA were synthesized from mRNA of the testis of mice and GS expression vectors were generated. The complete list of all primer sequences used in the study is provided as a Supplementary table. Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

We resolved cell proteins by SDS-PAGE before electro-blotting onto PVDF membranes. We incubated the membranes with the following primary antibodies overnight: anti-NCYM (1∶1000 dilution), anti-MYCN antibody (1∶1000 dilution; Calbiochem and Cell Signaling), anti-Lamin B (1∶1000 dilution; Calbiochem), anti-α-tubulin (1∶1000 dilution; Santa Cruz, CA, USA), anti-GST (1∶1000; Santa Cruz), anti-GSK3β (1∶1000 dilution; Cell Signaling), anti-phospho-GSK3β (S9) (1∶1000 dilution; Cell Signaling), anti-β-catenin (1∶1000 dilution; Cell Signaling), anti-phospho-AKT (S473) (1∶1000 dilution; Cell Signaling), anti-phospho-AKT (S308) (1∶1000 dilution; Cell Signaling), anti-AKT (1∶1000 dilution; Cell Signaling), anti-S6K (1∶1000 dilution; Cell Signaling), anti-phospho-S6K (T389) (1∶1000 dilution; Cell Signaling), and anti-actin (1∶4000 dilution; Sigma). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG at 1∶2000–1∶4000 dilution or anti-mouse IgG at 1∶2000 dilution; both from Cell Signaling Technology) and the bound proteins were visualized using a chemiluminescence-based detection kit (ECL and ECL pro kit, Amersham, Piscataway, NJ, USA; ImmunoStar LD, Wako).

PDE5-I vardenafil was obtained from Sequoia Research Products Ltd. (vardenafil citrate), Sigma Aldrich, or Selleckchem (vardenafil HCl). Nintedanib was obtained from Cayman Chemical. TGF-β1 was obtained from R&D Systems. The antibodies used in this study were as follows: fibronectin (Sigma Aldrich, F3648, St. Louis, MO, USA), CTGF (Santa Cruz Biotechnology, sc-14939, Dallas, TX, USA), αSMA (Sigma, A2547), p-SMAD3 (Cell Signaling 95205, Danvers, MA, USA), total SMAD3 (Abcam AB 28379), and GAPDH (Millipore MAB374). The secondary antibodies used for fibronectin ELISA included anti-rabbit IgG peroxidase (Sigma Aldrich, A0545) and anti-rabbit IgG-HRP (SantaCruz Biotechnology sc-2004). Additional antibodies for TGF-β1 signaling included the following: anti-mouse serpin E1/PAI-1 antibody (AF3828; R&D systems), anti-CTGF (sc-14939; SCBT), anti-αSMA (A2547; Sigma), anti-fibronectin (F3648, Sigma), anti-GAPDH (AB2302; Millipore, Burlington, MA, USA), anti-phospho-SMAD3 and anti-phospho-SMAD2 antibodies generated in our laboratory, anti-SMAD2 (ab63576; Abcam, Cambridge, UK), anti-SMAD3 (ab28379; Abcam), anti-phospho-AKT (Ser473; 9271, Cell Signaling), anti-phospho-AKT (T308; 4056, Cell Signaling), anti-phospho S6K (Thr389, 9234, Cell Signaling), anti-Akt (9272, Cell Signaling), and anti-S6K (9202, Cell Signaling).

Once whole cell or nuclear and non-nuclear protein fractions were obtained, the protein concentration in the supernatants was determined using Bradford reagent. Fifty micrograms of proteins was resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Blots were blocked and probed with various antibodies: anti-IGF1R β (#3027), anti-pIGF1R β (#3918), anti-AKT (#9272), anti-pAKT (#9271), anti-MAPK 42-44 (#9102), anti-pMAPK 42-44 (#9101), anti-S6K (#2708), anti-pS6K (# 9205), and anti-β actin (#4970) from Cell Signaling Technology (Boston, MA, USA); anti-lamin B (SC-6217) from Santa Cruz Inc. (Dallas, TX, USA); anti-pPDC subunit E1-α (NB110-93479) and ACSL5 (H00051703-M01) from Novus Biologicals (Centennial, CO, USA); anti-FAS (610962) from BD Biosciences (San Jose, CA, USA); and anti-GAPDH (MAB374) from Millipore (Darmstadt, Germany).

Cells were washed with ice-cold phosphate-buffered saline (PBS) and then solubilized with protease inhibitor (Upstate Biotechnology, Temesula, CA, USA)-containing RIPA buffer. Lysates were immunoblotted with indicated antibodies, anti-G9a, anti-DUSP4, anti-p-AMPK, anti-AMPK, anti-S6K and anti-p-S6K (T389) (Cell Signaling Technology), anti-H3K9me1 (Millipore, MA, USA), anti-GLP, anti-H3K9me2, anti-histone 3, anti-LC3B (Abcam), anti-H3K9me3, anti-caspase 3, anti-PARP, anti-p62, anti-β-actin anti-GAPDH, anti-α-tubulin (GeneTex), anti-p-Akt, anti-Akt, anti-p-ERK and anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA).

Erlotinib was kindly provided by F. Hoffman-La Roche Ltd; TPX-0005 was from TP Therapeutics, Inc.; dasatinib was purchased from Bio Vision; sorafenib was from Toronto Research Chemicals Inc; the other inhibitors were from Selleck Chemicals. Anti-HER2 and anti-pHER2 antibodies were purchased from Upstate Biotechnology; anti-EGFR, anti-pEGFR, anti-pHER3, anti-MET anti-pMET, anti-ERK1/2, anti-pERK1/2, anti-AKT, anti-pAKT (T308 or S473), anti-STAT3, anti-pSTAT3, anti-PTEN, anti-AXL, anti-CDCP1, anti-pCDCP1, anti-SRC, anti-FYN, anti-LYN, anti-YES, anti-LCK, anti-pSFK (Y416), anti-β-catenin, anti-S6K, and anti-pS6K antibodies were from Cell Signaling Technology; anti-E-cadherin antibody was from BD biosciences; anti-HER3 antibody was from Santa Cruz Biotechnology; anti-β-actin antibody was from Abcam, Inc.; Anti-GAPDH antibody was from PROMEGA; α-tubulin antibody was sourced from Sigma-Aldrich.

Western blot analyses were performed as previously described^{26 ,28 (link)}. Briefly, cells were lysed and sonicated in RIPA buffer (Thermo Scientific, IL, USA). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoretic separation, protein bands were transferred to Millipore Immobilon-P membrane followed by immunoblotting with antibodies against molecules of interest. The following primary antibodies were used for immunoblotting: anti-Smad2 (Cell Signaling, MA, USA, 1:1000), anti-p-Smad2 (Cell Signaling, 1:1000), anti-Smad3 (Cell Signaling, 1:1000), anti-p-Smad3 (Cell Signaling, 1:1000), anti-Akt (Cell Signaling, 1:1000), anti-p-Akt (Thr308 and Ser473) (Cell Signaling, 1:1000), anti-S6K (Cell Signaling, 1:1000), anti-p-S6K (Cell Signaling, 1:1000), anti-α-SMA (SIGMA, 1:2000), anti-GAPDH (Santa cruz Biotechnology, CA, USA, 1:5000) and anti-HSP70 (Invitrogen, 1:1000) antibodies. Chemiluminescence detection was performed using the ECL reagent (Bio-Rad Laboratories. Inc., CA, USA). Signal intensities of bands were quantified with Image J software (NIH).