Protein Expression Analysis in UM Cells

UM cells were seeded at 2.5 × 10⁵ cells per well of a 6-well plate and left to adhere for 24 h. Cells were treated with DMSO or 20 μM of test compound for 1,2,5,8, or 24 h. Total protein was extracted from cells as described (18 (link)). PVDF membranes (MilliporeSigma, Burlington, MA, USA) were probed with primary antibodies (ERK: Santa Cruz [sc-514302], 1:1,000, phospho-ERK: Santa Cruz [sc-7383] 1:1,000, MITF: Proteintech [13092-1-AP] 1:1,000, Bcl-2: Proteintech [12789-1-AP] 1:1,000, COX-2: Proteintech [12375-1-AP] 1:500, Calpain-2: Abcam [ab39165] 1:1,000, β-actin: Santa Cruz [sc-47778], α-Tubulin: Santa Cruz, 1:1,000). Secondary antibodies were anti-mouse IgG HRP-linked (Cell Signaling [7076S] 1:1,000), or anti-rabbit IgG HRP-linked (Cell Signaling [7074S] 1:1,000). Signal was detected using enhanced chemiluminescence as per the manufacturer’s instructions (Pierce™ ECL Western Blotting Substrate, Thermo Fisher Scientific, Rockford, IL, USA).

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Slater K., Bosch R., Smith K.F., Jahangir C.A., Garcia-Mulero S., Rahman A., O’Connell F., Piulats J.M., O’Neill V., Horgan N., Coupland S.E., O’Sullivan J., Gallagher W.M., Villanueva A, & Kennedy B.N. (2023). 1,4-dihydroxy quininib modulates the secretome of uveal melanoma tumour explants and a marker of oxidative phosphorylation in a metastatic xenograft model. Frontiers in Medicine, 9, 1036322.

Publication 2022

PVDF membranes phospho-ERK β-actin α-Tubulin Pierce™ ECL Western Blotting Substrate

Actin Anti igg Antibodies Bcl 2 Calpain Cells Chemiluminescence Compound 20 Cox 2 Dmso Membranes Mitf Mouse Protein Pvdf Rabbit α tubulin

Corresponding Organization : University College Dublin

Other organizations : Institut d'Investigació Biomédica de Bellvitge, Institut Català d'Oncologia, Universitat de Barcelona, St. James's Hospital, Royal Victoria Eye and Ear Hospital, University of Liverpool, Bellvitge University Hospital

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Variable analysis

independent variables

20 μM of test compound

dependent variables

Total protein
Protein expression levels of ERK, phospho-ERK, MITF, Bcl-2, COX-2, Calpain-2, β-actin, and α-Tubulin

control variables

UM cells seeded at 2.5 × 10^5 cells per well of a 6-well plate
Cells left to adhere for 24 h

controls

DMSO (negative control)

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