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. 2020 May 26;27(11):3065–3081. doi: 10.1038/s41418-020-0561-9

Fig. 3. Beclin 1 inhibits MLKL oligomerisation in the necrosome complex.

Fig. 3

a HT-29 cells expressing shGFP, shBECN1, and shMLKL were treated with TBZ. After treatment, the cells were lysed using lysis buffer and then fractionated according to their molecular size by gel filtration chromatography. The samples were analysed by immunoblotting. b HT-29 cells were treated with 30 ng/mL TNFα, 1 μM Birinapant, and 30 μM Z-VAD-FMK (TBiriZ) in the presence or absence of 2 μM GSK'872 for 5 h. After treatment, the cell lysates were immunoprecipitated using anti-caspase-8 antibody and then analysed by immunoblotting. c HT-29 and TC-1 cells were transfected with the indicated siRNAs, and then treated with TBZ in the presence or absence of Nec-1 for the indicated times. After treatment, the cell lysates were lysed under non-reducing conditions to detect MLKL oligomerisation or under reducing conditions and then analysed by immunoblotting. d HT-29 cells were transfected with the indicated siRNAs for 48 h and then treated with TBZ in the presence or absence of Nec-1 for the indicated time points. After treatment, the cell lysates were lysed using Triton X-114 buffer to divide them into detergent and aqueous phases. Each lysate was analysed by immunoblotting.