S mg132

pAC5.1 vectors with CLIP-CRY and TIM-SNAP-HA (S-TIM without the N-terminal 19 residue extension of LS-TIM was used(Sandrelli et al., 2007 (link))) were delivered into s2 cells using Effectene reagent (Cat# 301425, Qiagen) or TransIT-Insect Transfection Reagent (Cat# MIR6100, Mirus) following modified manufacturer instruction. Briefly, 1000×10⁴ cells in 10 mL growth media with FBS were placed in a 100 mm culture dish (Cat# 353003, Corning) on day 1. About 20 hours later, 4 μg DNA in total (CRY variants: TIM mass ratio 1:1, except CRYΔ: TIM = 7:1) was used with Effectene transfection reagent or 10 μg DNA when using TransIT-Insect reagent (same CRY: TIM mass ratio as using Effectene). 72 hours after transfection, 50 μM (S)-MG132 (Cat# 10012628, Cayman Chemical) was added to the 10 mL cells. After a brief mixing period, 10 mL cells were evenly split into two 60 mm culture dishes (Cat# 353002, Corning) to ensure the same cell population for dark and light conditions. After 2 hours of incubation, light sample was placed on a LED light pad (Autograph PRO1200) with light filter sheets to only pass light with wavelength of 440 nm to 500 nm. Then cells were centrifuged at 500 ×g for 5 minutes, room temperature, washed once with chilled PBS and stored at −80 °C until lysis.