Fig. 6: p62 regulation of ATF2 nuclear target activation.
From: The scaffold protein p62 regulates adaptive thermogenesis through ATF2 nuclear target activation
Co-immunoprecipitation (IP) in HEK293T cells transfected with Flag-p62 wt, Flag-p62Δ69-251, or HA-ATF2, respectively, and stimulated with isoproterenol as indicated. Flag-IP was performed to pulldown p62. Western blot analysis of pulldown (upper image) and input fraction (lower image) (a). Western blot analysis of ATF2 and p-ATF2 upon p62 overexpression in primary brown adipocytes isolated from 8-wk-old male p62Δ69-251 mice (b). Nuclear/membrane and cytosolic protein level of ATF2, p-ATF2 (Thr69/71), GAPDH, Histone 3 (H3), and UCP1 in BAT of 12-wk-old male wt and p62Δ69-251 mice upon injection of norepinephrine (1 mg kg−1) for 20 min (c). Nuclear/membrane and cytosolic protein level of p38, p-p38, GAPDH, Histone 3 (H3) as well as total lysates showing UCP1 in BAT of male wt and p62Δ69-251 mice upon injection of norepinephrine (1 mg kg−1) for 20 min (d). Nuclear/membrane and cytosolic protein level of ATF2, p-ATF2 (Thr69/71), GAPDH, Histone 3 (H3) as well as total lysates showing UCP1 upon 4 h stimulation with 1 µM isoproterenol in immortalized brown adipocytes from 6- to 8-wk-old male p62flx/flx Ucp1-Cre− or p62flx/flx Ucp1-Cre+ mice (e). Nuclear/membrane and cytosolic protein level of p38, p-p38, ATF2, p-ATF2 (Thr69/71), GAPDH, Histone 3 (H3) as well as total lysates showing UCP1 upon 4 h stimulation with 10 µM isoproterenol in immortalized brown adipocytes from wt or p62−/− mice (f). Panel (a) represents a representative example of three independently performed studies, each yielding similar results. Panels (b), (c), (d), and (f) are representative examples of two independently performed studies, each yielding similar results.
