3 mbz

The S2 cells were pelleted, washed, and resuspended in 0.5 mL RIPA buffer supplemented with 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma-Aldrich, St. Louis, MO, USA, 68178, #10837091001), 1X protease inhibitor cocktail (EpiGentek, Farmingdale, NY, #R-1101), 10 mM 3-MBZ (3-methoxybenzylamine, Sigma-Aldrich, St. Louis, MO, USA, 68178, #159891), and 0.5 mM BZA (benzylamine, Sigma-Aldrich, St. Louis, MO, USA, 68178, #185701). The cell suspension was incubated on ice for 30 min, gently vortexed, sonicated for 6 cycles (10 s on/10 s off) (Bioruptor300, Diagenode, Denville, NJ, USA), and spun down at 13,000 rpm for 10 min. The supernatant was used for Western blot analyses. The total RNA was extracted from the washed and pelleted cells using the Quick-RNA Miniprep Kit (Cat. #R1054) as per the manufacturer’s protocol (Zymo Research, Irvine, CA, USA, 92614) and eluted in RNase-free water. The total RNA was submitted (Novogene, Sacramento, CA, USA, 95826) for ribosomal-depleted paired-end RNA sequencing.

A total of 10⁷ Drosophila melanogaster S2 cells were pelleted and resuspended in 0.5 mL of RIPA buffer, to which protease inhibitors were added: 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma, St Louis, MO, USA, #10837091001), 1× protease inhibitor cocktail (EpiGentek, Farmingdale, NY, USA, #R-1101), 10 mM 3-MBZ (3-Methoxybenzylamine, Sigma, #159891), and 0.5 mM BZA (Benzylamine, Sigma, #185701). Cell suspension was put on ice for 30 min, followed by 6 cycles of sonication (each cycle: 10 s on/10 s off) on a Bioruptor300 (Diagenode, Denville, NJ, USA). The cell lysate was cleared by spinning at 13,000 rpm for 10 min. The resultant supernatant was then used for co-immunoprecipitation and Western blot analyses.

Drosophila melanogaster S2 cells 10⁷ were resuspended in 0.5 ml of RIPA buffer with fresh added inhibitors: 1 mM PMSF (Phenylmethylsulfonyl fluoride, Sigma, St Louis, MO, #10837091001), 1xProtease inhibitor cocktail (EpiGentek, Farmingdale, NY, #R-1101), 10 mM 3-MBZ (3-Methoxybenzylamine, Sigma, #159891) and 0.5 mM BZA (Benzylamine, Sigma, #185701), incubated for 30 min on ice with mild vortexing, and then sonicated for 6 cycles (10 sec on/10 sec off (Bioruptor300, Diagenode, Denville, MJ) and spun down at 13000 rpm for 10 min. Supernatant was used for Western blot analyses.