TMT analysis of detergent-soluble and insoluble fractions from HEK293T cells in normal conditions and upon heat shock

HEK293T cells were cultured according to standard protocols and heat shocked at 42oC for 6 hours. Detergent-soluble and insoluble fractions from HEK293T cells were obtained following the procedures described before. Specifically, HEK293T cells were lysed in NP40 lysis buffer (Invitrogen #FNN0021) with protease inhibitors by gentle pipetting. The cell homogenate was centrifuged at 14000 rpm and the supernatant was retrieved (detergent-soluble fraction). The remaining pellet was washed twice in ice-cold NP40 lysis buffer and then resuspended in urea buffer (RIPA buffer with 8M urea). The samples were then centrifuged at 14000 rpm for 10 min to pellet any remaining cell debris. The resulting supernatant (detergent-insoluble fraction) was retrieved and analyzed as explained below.