Fig. 3: M1-Ubi of AGO2 is catalyzed by LUBAC, which is induced by hypoxia. | Nature Communications

Fig. 3: M1-Ubi of AGO2 is catalyzed by LUBAC, which is induced by hypoxia.

From: Hypoxia regulates overall mRNA homeostasis by inducing Met1-linked linear ubiquitination of AGO2 in cancer cells

Fig. 3

a LUBAC catalyzed M1-Ubi of AGO2. LUBAC related plasmids were transfected into 293T cells and subsequently performed by M1-SUB pull-down assay for detection of AGO2 M1-Ubi. b M1-Ubi of endogenous AGO2 was enhanced by LUBAC. 293T cells were co-transfected with components of LUBAC. Lysates were used for IP with anti-AGO2 antibody, followed by WB with a specific linear ubiquitin antibody 1E3 clone. c LUBAC catalyzed M1-Ubi of GST-AGO2 in vitro. Purified GST-AGO2 were incubated with lysates from 293T cells transfected with components of LUBAC, and followed by GST pull-down/WB assay with a specific linear ubiquitin antibody LUB9 clone. d LUBAC increased M1-Ubi of endogenous AGO2. Stable HeLa cells expressing HOIP and HOIL-1L were lysed for IP with anti-AGO2 antibody, and then determined M1-Ubi of AGO2 by WB with antibody 1E3 clone. e In vitro M1-Ubi assay for AGO2. Flag-AGO2 purified from 293T cells was co-incubated with purified ubiquitin and LUBAC proteins for in vitro M1-Ubi assay. f Knockdown of HOIP inhibited AGO2 M1-Ubi. Lysates from stable HeLa-shHOIP or -pLKO.1 cells were used for IP/WB. g AGO2 M1-Ubi was dependent on the catalytic activity of HOIP, but not that of HOIL-1L. 293T cells were co-transfected with indicated plasmids, and M1-Ubi of AGO2 was detected by IP/WB. h M1-Ubi of AGO2 was removed by OTULIN. 293T cells were co-transfected with indicated plasmids, and M1-Ubi of AGO2 was analyzed by M1-SUB pull-down/WB. i Knockdown of OTULIN increased AGO2 M1-Ubi. Stable HeLa-shOTULIN cells were transfected with indicated plasmids, and AGO2 M1-Ubi was analyzed by M1-SUB pull-down/WB. j OTULIN mutants (W96A, C129A) did not inhibit AGO2 M1-Ubi. OTULIN or indicated OTULIN mutants were transfected into 293T cells, respectively. M1-Ubi of AGO2 was analyzed by IP/WB. k K820 is a major site for M1-Ubi of AGO2. The M1-Ubi of AGO2-WT and indicated AGO2 mutants were detected by M1-SUB pull-down and WB. l Hypoxia-induced M1-Ubi of AGO2. 293T and HeLa cells were treated with hypoxia or CoCl2 (300 μM) for indicated times, and AGO2 M1-Ubi was determined by IP/WB.

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