Fig. 2: Hypoxia promotes the interaction of AGO2 with LUBAC.

a Scatter distribution plot of proteins interacting with AGO2 under hypoxia and normoxia conditions were analyzed by mass spectrometry (MS). HeLa cells expressing Flag-AGO2 were treated with hypoxia or normoxia for 24 h, respectively. Cell lysates were used for co-IP with anti-Flag antibody, and followed by MS analysis. b, c Ectopically expressed Flag-HOIP or Flag-HOIL-1L interacted with AGO2. Myc-AGO2 was co-transfected with Flag-HOIP (b) or Flag-HOIL-1L (c) into 293T cells, the association of AGO2 with HOIP or HOIL-1L was determined by co-IP/WB. d HOIP, HOIL-1L and SHARPIN interacted with AGO2. Lysates from HeLa cells were used by co-IP with anti-AGO2 antibody, and followed by WB. e SHARPIN associated strongly with HOIP, HOIL-1L but weakly with AGO2. Lysates form HeLa cells were used by co-IP with anti-SHARPIN antibody, and followed by WB. f Hypoxia promoted the interaction of AGO2 with HOIL-1L and HOIP. HeLa cells treated with hypoxia were lysed for co-IP with anti-AGO2 antibody, and followed by WB with anti-HOIP and anti-HOIL-1L antibodies. g Increasing expression of HOIL-1L promoted AGO2 recruiting more LUBAC. Flag-HOIL-1L was transfected in a dose-increasing manner with HA-AGO2 into 293T cells. co-IP was performed with anti-HA antibody, and then endogenous HOIP and Flag-HOIL-1L bound to AGO2 were conducted by WB. h Knockdown of HOIL-1L decreased the interaction of AGO2 with HOIP. Stable HOIL-1L-knockdown HeLa cells were used for co-IP/WB with indicated antibodies. i AGO2 prolyl-4-hydroxylation did not influence its interaction with HOIL-1L and HOIP. 293T cells transfected with Flag-AGO2-WT or prolyl-4-hydroxylation mutant Flag-AGO2-P700A were treated with hypoxia for indicated times, and then lysed for co-IP/WB with indicated antibodies. j Hypoxia enhanced the association of HOIL-1L with HOIP and AGO2. HeLa cells treated with hypoxia for indicated times were lysed for co-IP/WB with indicated antibodies. k, l Analyses of HOIP and HOIL-1L binding domains with AGO2. Various truncated forms of Flag-HOIP (k) or Flag-HOIL-1L (l) were individually co-transfected with HA-AGO2 into 293T cells, and then lysates were used by co-IP with anti-HA antibody, followed by WB.