Aldrithiol 4

In vivo crosslinking was adapted from a previous publication (51 ). Briefly, HEK-STIM1/2^−/−/UNC93B1^−/− cells were grown and transfected with plasmids expressing STIM1 A230C, M241C, or L251C with pcDNA3 or pcDNA3 expressing UNC93B1-FLAG-MYC in a 6-well plate format. On the day of experiment, cells were treated or not with 0.5 μM Tg for 5 min and immediately incubated with 180 μM aldrithiol-4 (catalog no.: 143057; Sigma) for 20 min at 37 °C. Cells were then washed twice with cold PBS and lysed on ice for 15 min with 200 μl/well of TUNES buffer (100 mM Tris [pH 7.2], 6 M urea, 10 mM EDTA, 1% SDS, 0.4 M NaCl, and 10% glycerol) containing 50 mM N-ethylmaleimide (catalog no.: E1271; Sigma) and protease and phosphatase inhibitors. Cells were then scraped and sonicated with the Covaris S220 Focused-Ultrasonicator (Power 140 W, duty factor 10%, cycles per burst 200, 44 s) at the iGE3 Genomics platform in UNIGE. About 1% of bromophenol blue were added to the sonicated samples and prepared for Western blot analysis.

The kinetic parameters of WT AAC(3)-IIIa against a panel of aminoglycosides (gentamicin, kanamycin, sisomicin, tobramycin, plazomicin, neomycin, paromomycin, and ribostamycin) were obtained using the ThermoFisher NanoDrop One^C Spectrometer. AAC(3)-IIIa mutants were run against solely gentamicin. The acetylation of aminoglycosides was measured by a coupled assay, where the formation of pyridine-4-thiolate can be detected at 324 nm [13 (link),14 (link)]. The kinetic assays were performed at room temperature (22°C) in quartz cuvettes (pathlength 1 cm) at a final volume of 0.8 ml. Reaction solution contained 25 mM MOPS pH 6.5, 100 mM NaCl, 500 μM 4,4’-dipyridyl disulfide (Aldrithiol™-4, Sigma-Aldrich), 150 μM acetyl coenzyme A, and aminoglycoside concentrations varying from 1.25 to 160 μM. The reaction was initialized by the addition of AAC(3)-IIIa (WT and mutants) to a final concentration of 0.3 to 0.5 μM, where UV absorbance was measured over 5 minutes. All reactions were run in triplicate, and data analysis was performed using the GraphPad5 software.