Precise tris glycine gel

M-PER Mammalian Protein Extraction Reagent (Cat#78503, Thermo Fisher Scientific, Waltham, MA) and Proteinase Inhibitor Cocktail (Cat#P8340, Sigma-Aldrich, St. Louis, MO) were used to extract proteins from U87_TRAF3IP2KD, U87_{Control shRNA}, U118_TRAF3IP2KD, and U118_{Control shRNA} cells. After gel electrophoresis of equal amounts of protein using 12% Precise Tris–Glycine Gels (Cat#0025267, Thermo Fisher Scientific), Laemmli sample buffer (Cat#161-0747, Bio-Rad Laboratories, Hercules, CA), and BenchMark Pre-Stained Protein Ladder (Cat#10748-010, Invitrogen, Carlsbad, CA), the proteins were electroblotted and the following primary antibodies were used: GAPDH (0.0002 mg/ml; Cat#ab9485, Abcam, Cambridge, MA), TRAF3IP2 (0.01 mg/ml; Cat#WH0010758M1-100UG, Sigma-Aldrich), CD31 (0.01 mg/ml; Cat#PA5-16301, Invitrogen), IL1β (0.01 mg/ml; Cat#710331, Invitrogen), IL6 (0.01 mg/ml; Cat# MA5-23698, Invitrogen), IL8 (0.01 mg/ml; Cat# PA5-86028, Invitrogen). Goat Anti-Rabbit IgG-HRP (Cat#sc-2004, Santa Cruz Biotechnology, Inc.) or Donkey Anti-Mouse IgG-HRP (Cat#sc-2318, Santa Cruz Biotechnology, Inc.) served as secondary antibodies.

MCF7 cells were seeded to 60-80 % confluency in 60 mm dishes and serum starved 24 h prior to the indicated treatments. Following treatment, cells were washed twice with cold PBS and scraped into lysis buffer: RIPA buffer containing NP-40 supplemented with sodium fluoride (50 mM), sodium orthovanadate (1 mM), phenylmethylsulfonylfluoride (1 mM), 0.1 % SDS, 0.5 % sodium deoxycholate and protease cocktail (1×). Cell lysate protein concentration was determined by Bradford protein assay (Bio-Rad). Equal protein concentrations per lysate were loaded on a 4-20 % Precise Tris-Glycine Gels (Thermo Scientific) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked in 5 % Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad) for 1 h at room temperature and then incubated with primary antibodies (1:500 for ERα; 1:3000 for GPER; 1:10,000 for actin) in 3 % BSA overnight at 4 °C. The blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000) or goat anti-mouse IgG for actin (1:5000) in 3 % BSA for 1 h at room temperature and developed using Supersignal West Pico Chemiluminescent Substrate (Thermo Fisher). Films were scanned and quantified using ImageJ software (National Institutes of Health).

Isolated melanosomal fractions were introduced to 1% Triton-X-100 buffer containing inhibitors (PMSF and protease inhibitor cocktail, Sigma-Aldrich) and disrupted with sonication. Twenty μg of protein was loaded on each well of SDS-PAGE gel (4–20% Precise Tris-Glycine Gels, Thermo Fisher Scientific Inc. Waltham, MA, USA). Proteins were transferred onto PVDF membrane (Bio-Rad) with semi-dry electroblotting (Trans-Blot^® SD Semi-Dry Transfer Cell, Bio-Rad). Primary antibody incubations were conducted at +4°C overnight (mouse-anti HSP60 1:1000, #ADI-SPA-806-F, Enzo Life Sciences Inc., Farmingdale, New York, USA; Anti-Na⁺/K⁺ATPase α1, 1:1000, 05–369, Merck Millipore, Merck KGaA, Darmstadt, Germany; Atp6v0a1 antibody, 1:500, GTX44653, GeneTex Inc., Irvine, CA, USA, Rab27a antibody 1:500, 0023, SICGEN–Research and Development in Biotechnology Ltd, Cantanhede, Portugal). Secondary antibody incubations (goat anti-mouse, sc-2005 and goat anti-rabbit sc-2030, 1:10 000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) took place at room temperature for 45 min. Protein-antibody-complexes were detected with chemiluminescence reaction (Image Quant RT ECL, GE Healthcare, Little Chalfont, UK) using Amersham^™ ECL^™ Prime Western Blotting Detection Reagent (GE Healthcare).