Fig. 7: OPTN inhibits the dimerization of JAK2 and subsequent STAT3 activation.
a HA-OPTN expression plasmid was transfected with Flag-JAK1, Flag-JAK2, or Flag-JAK3 expression plasmid into HEK293 cells. Expression of OPTN and JAKs was confirmed by immunoblotting. Interaction between OPTN and JAKs was determined by immunoprecipitation (IP) with anti-HA antibody followed by immunoblotting with anti-Flag antibody. b Interaction between endogenous OPTN and JAK2 in BMDCs was analyzed by IP. c Schematic illustration of the truncated JAK2. Interactions between OPTN and truncation JAK2 in transiently transfected HEK293 cells were determined by IP. d Interactions between JAK2 with OPTN or OPTNΔUBD in transiently transfected HEK293 cells were determined by IP. e Interactions between HA-JAK2 and FLAG-JAK2 in transiently transfected HEK293 cells was determined by IP. f, g HEK293 cells were transfected with FLAG-JAK2 and HA-OPTN or empty vector (f). BMDCs were transfected with HA-OPTN or empty vector (g). Whole-cell lysates were incubated with disuccinimidyl suberate (DSS). JAK2 antibody marked two bands: the upper band referring to the dimer and the lower band representing the monomer of JAK2. h Interaction of FLAG-JAK2 and HA-STAT3 in transiently transfected HEK293 cells was determined by IP. i BMDCs were transfected with HA-OPTN or empty vector. Interaction between endogenous STAT3 and JAK2 in BMDCs was analyzed by IP. j ChIP-seq analysis of the binding between STAT3 and Il-10 or Il-6 from GSE27161. k Il-10 and Il-6 mRNA level in Ctrl and Optn deficient BMDCs stimulated with LPS for different times. n = 3 independent experiments, P(4) = 3.2E−06, P(8) = 0.0497, P(12) = 1.2E−05, P(16) = 3.9E−07, P(24) = 0.0060 of Il-10. Data in (a–i) are representative of three independent experiments. Data are presented as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Unpaired two-tailed Student’s t-test. Source data are provided in Source data file.
