Anti elk1

Samples for Western blots were collected, prepared, and analyzed as described previously [31 (link),32 (link)]. 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 sulfatepolyacrylamide 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 horseradish peroxidase (HRP)-linked secondary antibody (1:1000, #7074S, Cell Signaling, Beverly, MA, USA) 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 Image J (National Institutes of Health; NIH, Bethesda, MA, USA). The primary antibodies used in this study were anti-ELK1 (1:500, #9182S, Cell Signaling) and anti-β-actin (1:2000, #8457L, Cell Signaling). The band intensities of ELK1 were normalized to those of β-actin.

Anti-MDM4 antibodies were purchased from Bethyl Laboratories. Anti-MDM2 antibody was purchased from R&D Systems. Anti–acetylated p53, anti-p53, anti–phospho ELK1, anti-ELK1, and anti–lamin B antibodies were from Cell Signaling. Anti-αSMA antibody was from American Research Products. Anti-Fas (CH11), anti-DD1α, anti-CD68, and anti–collagen I antibodies were from Thermo Fisher Scientific. Anti-AGE antibody was from Abcam. Anti-fibronectin and anti-GAPDH antibodies were from Santa Cruz Biotechnology. CXCL10 neutralizing antibody was from Thermo Fisher Scientific. CX3CL1 neutralizing antibody was from R&D Systems. The activities of commercial CXCL10 and CX3CL1 neutralizing antibodies were verified by functional blocking of cell chemotaxis. C646 and tamoxifen were from Sigma. CA was from MedChem Express. Validated MDM4 siRNAs were from OriGene.

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.