Anti nf bp65

Samples for Western blots were collected, prepared and analysed as described previously.³⁸ Briefly, 661W cells were harvested and lysed in a Tris lysis buffer (in mM): 50 Tris, 1 EGTA, 150 NaCl, 1% Triton X‐100, 1% β‐mercaptoethanol, 50 NaF, and 1 Na₃VO₄, pH 7.5. Samples were separated on 10% sodium dodecyl sulphate‐polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked in 3% BSA in Tris‐buffered saline, Tween 20 (TBST) at room temperature for 1 h and incubated in primary antibodies overnight at 4°C. After washing with TBST, the membranes were incubated in anti‐rabbit IgG HRP‐linked secondary antibody (1:1000, #7074S, Cell Signaling) at room temperature for 1 h. The blots were visualized using Super Signal West Pico/Femto chemiluminescent substrate (#34078 / #34096, ThemoFisher). Band intensities were quantified using ImageJ (NIH). The primary antibodies used in this study were as follows: anti‐ELK1 (1:500, #9182S, Cell Signaling), anti‐phospho NFĸB P65 (1:1000, #3033, Cell Signaling), anti‐NFĸB P65 (1:1000, #8242S, Cell Signaling) and anti‐β‐actin (1:2000, #8457L, Cell Signaling). The band intensities of ELK1 were normalized to those of β‐actin. The band intensities of phosphorylated NFĸB P65 (pP65) were normalized to those of total NFĸB P65 (P65) and β‐actin.

Western blottings were performed as described [45 (link)]. Antibodies used included anti-phospho-β-Catenin (Ser33/Ser37/Thr41), anti-GSK-3β, anti-Axin-2, anti-phospho-JNK, anti-NF-ĸB/p65, anti-phospho-NF-ĸB/p65 and anti-IĸBα, which were purchased from Cell Signaling Technology, anti-Cyclin D1 (Santa Cruz) and anti-GAPDH antibody (Abcam). Specific bands were visualized with the West Dura Extended Duration Substrate (Pierce) and Chemiluminescence Analysis System (ChemiScope Series, Clinx). The cropped blots were displayed in the figures. The intensities of bands were measured by the ChemiScope Analysis System.