Gtx118000

MIA-PaCa 2 cells were seeded in a 6-well tissue culture plate (cell density 1.5 × 10⁶/well) in DMEM medium supplemented with 10% foetal bovine serum, 1% L-glutamine, and 1% penicillin and streptomycin at 37 °C and 5% CO₂. Cells were serum-starved for 24 h and then incubated with LIF (10 ng/mL) alone or plus mifepristone (10, 20, 50 µM) for 10 min. Total lysates were prepared by homogenization of MIA-PaCa2 cells in Ripa buffer containing phosphatase and protease inhibitors. Protein extracts were electrophoresed on 12% acrylamide Tris-Glycine gel (Invitrogen), blotted to the nitrocellulose membrane, and then incubated overnight with primary Abs against STAT3 (sc-8019 1:500; Santa Cruz Biotechnology) and phosho-Stat3 (GTX118000 1:1000; Genetex). Primary Abs were detected with the HRP-labelled secondary Abs. Proteins were visualized by Immobilon Western Chemiluminescent Reagent (MilliporeSigma) and iBright Imaging Systems (Invitrogen). Quantitative densitometry analysis was performed using ImageJ software. The degree of STAT3 phosphorylation was calculated as the ratio between the densitometry readings of p-STAT3/STAT3.

MK45 cells were seeded in six-well tissue culture plate (cell density, 400 × 10³ per well) in 100 µl of RPMI 1640 medium supplemented with 10% FBS, 1% l-glutamine, and 1% penicillin and streptomycin at 37°C and 5% CO₂. Cells were serum-starved for 24 h and then incubated with LIF (10 ng/ml) and EC359 (25 and 100 nM), alone or in combination, for 48 h. Total lysates were prepared by homogenization of MKN45 cells in Ripa buffer containing phosphatase and protease inhibitors. Protein extracts were electrophoresed on 12% acrylamide Tris-Glycine gel (Invitrogen), blotted to nitrocellulose membrane, and then incubated overnight with primary Abs against Jak1 (1:500; sc-7228, Santa Cruz Biotechnology), phospho-Jak1 (1:1,000; GTX25493, GeneTex), STAT3 (1:500; sc-8019, Santa Cruz Biotechnology), phosho-Stat3 (1:1,000; GTX118000, GeneTex), and Gapdh (1:1,000; bs2188R, Bioss Antibodies). Primary Abs were detected with the HRP (horseradish peroxidase)-labeled secondary Abs. Proteins were visualized by Immobilon Western Chemiluminescent Reagent (MilliporeSigma) and iBright Imaging Systems (Invitrogen). Quantitative densitometry analysis was performed using ImageJ software. The degree of JAK1 and STAT3 phosphorylation was calculated as the ratio between the densitometry readings of p-Jak1/Jak1 and p-STAT3/STAT3, respectively.