3.5. Western blot analysis of p-STAT3

After 24 h, B16.F10 cells harvested by trypsin/EDTA and washed twice with ice-cold PBS and transferred to Eppendorf tubes imbedded in ice bucket. Cells were, then, lysed using lysis buffer containing 2 mM Na₃VO₄, 30 mM HEPES (pH 7.5), 2 mM EGTA, 2% NP-40, 0.5 mM dithiothreitol (DTT), and 1:100 protease/phosphatase inhibitor cocktail. Cell lysates were centrifuged for 20 sec at 16,000g. Thereafter, NaCl was added to samples to final concentration of 420 mM, cell lysates were centrifuged for 20 min at 16,000g. Supernatant was then transferred to new Eppendorf tubes, where and pellets were discarded and protein extract was determined using Micro BCA™ Protein Assay Kit. Thereafter, equal amounts of protein (20 μg) were mixed with equal volumes of loading buffer (0.5 M Tris-HCl pH 6.8, 20% glycerol, 10% SDS, 1.5% bromophenol blue, 5% 2-mercaptoethanol). Then, samples were dipped in boiling water for 5 min and then loaded on 10% SDS-PAGE gel. Electrophoresis was then conducted in Tris-Glycine SDS Running Buffer for 90 min. Proteins were then transferred into activated PVDF membrane using Bolt® Transfer Buffer containing methanol at 140 V for 2 h. Thereafter, the membrane was incubated overnight at 4 °C with Blocker BSA solution containing 0.1% (v/v) TWEEN® 20 that was also used to thoroughly wash membranes, where they are probed with antiphosphotyrosine (Y⁷⁰⁵) STAT3 mAb (1:500) for 2 h. The membrane was then washed 5 times under gentle shaking. Then, goat antimouse polyclonal Ab (1:50,000) was added to the membranes in blocking buffer and kept at room temperature for 90 min under gentle shaking. Thereafter, the membrane was further 5 times before development using Amersham ECL Prime Western Blotting Detection Reagent kit. The membranes were then stripped, washed, blocked, and probed with anti-actin Ab (I-19) (1:1000) dilution and developed as previously described.