Tip sonicator

Cells were collected by scraping in 250 μL ice cold 1x RIPA buffer with protease inhibitor. Lysates were sonicated using a tip sonicator (Branson) followed by clearing of insoluble material by centrifugation. The protein concentration of the supernatants was estimated by absorbance measurements on a Nanodrop instrument. 30 μg of lysate per lane was resolved using 4–12% Bis-Tris SDS PAGE gels (Invitrogen) and then transferred to PVDF membrane (Bio-Rad). Membranes were blocked in 5% non-fat dry milk (Biorad) in TBST (Tris-buffered saline with 0.1% Tween 20) for 1 hr at room temperature. Primary antibodies were incubated in milk in TBST overnight at 4 °C. Primary antibodies used here were anti-TUBG1 (Sigma, T6557, 1:2000), FLAG (Sigma, M2, 1:2000), anti-NRF2 (Proteintech, 66504, 1:2000), anti-succinyllysine (PTM Biolabs, PTM-401, 1:1000), anti-MYC (and anti-SUCLG2 (Bethyl Laboratories, A305-533A, 1:2000). After 5x TBST washes for 15–30 minutes, fluorophore-conjugated (1:2000, LI-COR) or HRP-conjugated (1:3000, ThermoFisher) secondary antibodies were incubated in milk for 1 hr and then washed again with TBST before visualizing on a LI-COR Odyssey fluorescent scanner or with film.

Stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), and cholesterol (Chol) from ovine wool were purchased from Avanti Polar Lipids (Birmingham, AL) and used without further purification. Tris salt and potassium chloride (KCl) were purchased from Merck (Germany). Lipid stock solutions of POPC and POPS in chloroform were mixed in appropriate molar ratios, dried under a stream of nitrogen, and placed under vacuum for 1 h. After drying the lipid films were rehydrated in 10 mm Tris, 100 mm KCl solution and vortexed for 5 min. SUVs were prepared by sonicating the rehydrated liposome solution for 40 min using a Branson tip sonicator. Thereafter, the SUVs were centrifuged at 16,100 × g to remove any tip residue from the sonicator probe. For preparation of LUVs, the rehydrated liposome solution (after the vortexing step) was subjected to multiple cycles of freeze-thawing in liquid nitrogen until the resulting solution was clear. Thereafter, the solution was extruded through a polycarbonate membrane of pore size 100 nm. The SUVs and LUVs were used immediately after preparation.