Anti pstat3 s727

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.

Total protein extracts were obtained by homogenization of pools of 20 6-dpf larvae in ice cold RIPA buffer (ThermoFisher, 89900) and Complete EDTA-free protease inhibitor cocktail (Sigma, 11873580001). For western blot analysis 40 μg of protein extracts were loaded per well on Bolt 4–12% Bis-Tris Plus Gels (ThermoFisher, NW04120BOX) and blotted on PVDF immobilon-p membranes (Millipore, IPFL00010). Dried membranes were then washed with PBS (Sigma, P4417) with 0.1% (w/v) Tween20 and incubated overnight with primary antibodies at 4 °C: anti-HIF1α (1:500, MA1-16504 Invitrogen); anti-STAT3 (1:1000, 9139 S Cell Singaling); anti-pSTAT3 Y705 (1:1000, D3A7 Cell Signaling), anti-pSTAT3 S727 (1:1000, 9134 Cell Signaling) and anti-βActin (1:5000, MA1-744 ThermoFisher). Secondary anti-Rabbit and anti-Mouse HRP-conjugated antibodies (1:5000, 170-6515 BIORAD, and 1:5000, 170-6516 BIORAD, respectively) were incubated for 1 h at room temperature and protein bands detected by chemiluminescence on an Alliance MINI HD 9 Blot Imaging System. Quantification of the signal was performed with ImageJ.

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.

The protein was separated by SDS-PAGE and transferred on nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Western blot was performed by SNAP i.d. protein detection system (Millipore). The hybridized bands were detected by enhanced chemiluminescence (ECL) detection kit (Thermo Scientific brand of Thermo Fisher Scientific, Inc.). The antibodies were as follows: anti-AMPK, anti-pAMPK, anti-mTOR, anti-pmTOR, anti-pSTAT3 Y705, anti-pSTAT3 S727, and anti-STAT3 (all from Cell Signaling); TRAF6 (Santa Cruz); and β-actin (Sigma).

Murine splenocytes were treated with IL-6 (20 ng/mL), with or without fraxinellone for 30 min. At the given time, total cellular proteins from cells were extracted using RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo scientific, Waltham, MA, USA). Polyacrylamide gel electrophoresis was performed at 100 V for 1.5 h, and proteins were transferred to polyvynilidene fluoride membrane (Bio-Rad, Hercules, CA, USA). To evaluate protein expression, membranes were incubated with the following antibodies: anti-STAT3, anti-phospho-STAT3_Y705 (pSTAT3_Y705), anti-pSTAT3_S727 (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin antibodies (Sigma-Aldrich). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Thermo Scientific) or goat anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA). Reactive signals were evaluated using SuperSignal^® West Pico Chemiluminescent substrate (Thermo Scientific), and the membranes were then exposed to an Amersham Imager 600 (GE Healthcare Bioscience, Pittsburgh, PA, USA).

LNCaP and C4–2 cells were treated with GPB730 and enzalutamide for 72 h. Cell lysates were prepared and western blot analysis was performed according to previous publication [21 (link),22 (link)]. Primary antibodies used were anti-STAT3 (#4904 Cell Signaling Technology), anti-pSTAT3-T705 (ab76315 Abcam), anti-pSTAT3-S727 (#9134 Cell Signaling Technology), c-myc (ab32072 Abcam), survivin (#2808 Cell Signaling Technology), PSA (sc7638 Santa Cruz) and androgen receptor (ab108341 Abcam). Beta-actin (#A5441 Sigma) was used as loading control. LNCaP cells stimulated with 50 ng/ml IL-6 for 30 min and DU145 cells were used as positive controls for pSTAT3-S727 and pSTAT3-T705 expression.