Win 62 577

L. reuteri DSM 17938 (Biogaia AB, Stockholm, Sweden) was purchased from Aché Pharmaceutical Laboratories (São Paulo, Brazil). Ethanol, Substance P acetate salt hydrate, Resiniferatoxin (RTX), and non-peptide NK₁ tachykinin receptor antagonist (WIN 62,577) were purchased from Sigma Aldrich (St. Louis, MO, USA). The other reagents were of analytical grade bought from standard commercial companies. One milligram of RTX was dissolved in 1 mL of 95% ethanol and stored at −20 °C. When needed, this solution was diluted in 0.9% saline to the necessary concentration. WIN 62,577 was dissolved in dimethyl sulfoxide (DMSO). The other compounds were dissolved in saline when needed.

U2OS and HeLa cells were treated for 3 h with 1 µm TAK243, 10 µm MG132, 20 µm PR619, 20 µM WIN 62,577 (Sigma, Cat: W104) or DMSO. 250 µM Me₄BodipyFLAhx₃Leu₃VS was added 10 min before harvesting cells by washing twice with ice-cold PBS and lysing in RIPA lysis buffer (25 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, EDTA-free protease inhibitor)^{76 (link)}. Samples were equalised and prepared for gel electrophoresis as described above. Samples were run on 12% Bis-Tris gels (ThermoFisher Scientific, Cat: NP0341PK2) and fluorescence was measured using a Typhoon FLA 9500 (GE Healthcare) using a 473 nm laser (Rho) and emission filters of 530 nm (Rho). The gels were then used for WB and stained for ubiquitin as described above.