Anti pstat3 y705

Primary antibodies used included rabbit anti-Ras (#3965, Cell Signaling Technologies, 1:1000), anti-pSTAT3 Y705 (#9145 S, Cell Signaling Technologies, dilution 1:1000), anti-pSTAT3 S727 (#9134D, Cell Signaling Technologies, dilution 1:1000), anti-STAT3 (#4904 S, Cell Signaling Technologies, dilution 1:1000), mouse anti-SV40-T antigens (MABF121, EMD Millipore, dilution 1:1000), and mouse anti-β-actin (A5441, Sigma-Aldrich, 1:5000). For IL-6 signaling neutralization studies, anti-IL-6R antibody (MAB227, R&D Systems) or control IgG (sc-2025, Santa Cruz Biotechnology) at 2 µg/ml concentration was added to CM for 1 h prior to adding CM to epithelial cells. Cells were treated with CM for 2 h prior to harvesting cells for western blotting. Anti-mouse (#7076, dilution 1:10000), and anti-rabbit (#7074, dilution 1:10000) horseradish peroxidase (HRP) linked secondary antibodies were purchased from Cell Signaling Technologies. Cell lysates were prepared in radioimmunoassay buffer and analyzed by western blotting^{43 (link)}. Uncropped blots are presented in the Source data file.

Immunoblotting and immunoprecipitation were performed as previously described^{52 (link)}. Anti-MSK1, anti-Aurora B, anti-β-actin, anti-NFATc2, anti-GADPH, anti-α-tubulin, anti-laminA/C were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-H3S10 and anti-H3 were from Active Motif (Carlsbad, CA, USA). Anti-STAT3, anti-AKT, anti-p42/44, anti-p-STAT3 Y705, anti-p-STAT3 S727, anti-p-AKT S483, anti-p-p42/44 were from Cell Signaling Technology (Danvers, MA, USA).

Western blot analyses were performed as described previously^{6 (link)}, using the following primary antibodies: anti-PLOD3 (Proteintech Group, Inc., Chicago, IL, USA); anti-HA, anti-ERK1/2, anti-p38, anti-K-RAS, anti-H-RAS (Santa Cruz Biotechnology Inc.); anti-p-anti-ERK1/2, anti-p-p38, anti-p-JNK, anti-JNK, anti-STAT3, anti-p-STAT3 (Y705), and anti-p-STAT3 (S727) (Cell Signaling Technology Inc.) antibodies. β-actin (Sigma) was used as a loading control.

For Western blots, whole-cell extracts were prepared using the radioimmunoprecipitation assay (RIPA) lysis buffer (LPS solution, Daejeon, Korea) comprised of 150 mM NaCl, 1% NP-40, 0.1% SDS, and 50 mM Tris (pH 7.4) containing 1 mM NaF, 1 mM Na₃VO₄, 1 mM ß-glycerophosphate, 2.5 mM sodium pyrophosphate, and protease inhibitor cocktail (Roche, Basel, Switzerland). Proteins were quantified using the Bradford assay reagent (Bio-Rad) according to the manufacturer’s instructions. Proteins (20–30 μg) were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting was performed as described above. The primary antibodies used were as follows: Anti-pSTAT3 (Y705) (1:500, 9145S; Cell Signaling Technology, Danvers, MA, USA), anti-STAT3 (1:1000, 4904S; Cell Signaling Technology), anti-fibronectin (1:500, ab6328; Abcam, Cambridge, UK), anti-integrin α5 (1:1000, ab150361; Abcam), anti-JAK1 (1:500, 50996S; Cell Signaling Technology), anti-JAK2 (1:1000, 3230S; Cell Signaling Technology), anti-GP130 (1:2000, ab226346; Abcam), anti-LDB1 (1:1000, ab96799; Abcam), anti-LMO2 (1:500, NB110-78626SS; Novus Biologicals, Littleton, CO, USA), and anti-α-tubulin (1:10,000, T6199; Sigma Aldrich). α-Tubulin was used as a loading control.

Anti-PAK1, anti-pStat3 (Y705), and anti-JAK2 antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-p65, anti-Stat3, anti-β-actin, and anti-Lamin b were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-CD44-FITC and anti-CD24-PE antibodies were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Antibodies for ChIP and EMSA experiments were Anti-PAK1 and anti-pStat3. Human PAK1 siRNA and scrambled siRNA were purchased from Bioneer (Daejeon, Korea). The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Whole-cell extracts from DCs or CAFs were prepared in RIPA buffer (Cell Signaling, Boston, MA, USA) plus Complete Protease and Phosphatase Inhibitor Cocktail (ThermoFisher, cat.no. 78440). Total cell-associated proteins were separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a PVDF membrane. The membrane was blocked with 1% BSA in tris buffered saline, 0.1% Tween 20 (TBS-T) for 2h at room temperature, and then incubated (overnight, 4°C) with primary antibodies (anti-GAPGH, cat. no. 5174; anti-STAT3, cat. no. 4904; anti-p-STAT3 (S727), cat.no. 34911; anti-p-STAT3 (Y705), cat-no. 9145; anti-NF-κB/p65, cat.no. 8242; anti-p-NF-κB/p65, cat.no 3033; Cell Signaling; anti-TDO2, cat.no. ab76859) diluted 1:1000 (in TBS-T with 1% BSA). Subsequently, the membrane was washed 5x in TBS-T and then incubated with an anti-rabbit or anti-mouse HRP-conjugated secondary antibody (diluted 1:2000; Cell Signaling) for 1h at room temperature. Finally, proteins transferred to the membrane were visualized with Enhanced Chemiluminescence at ImageQuant LAS 4000 CCD (GE Healthcare Bio-Sciences, PA, USA). Relative intensity was assessed using ImageJ software.