Fig. 5
STAT6 interacts with Rheb and inhibits mTOR signaling under hypoxic conditions. a Schematic of inhibition of mTOR signaling under hypoxic conditions. b C U87MG cells transfected with non-targeting control siRNA (CONsi) or pooled siRNA targeting STAT6 (STAT6si) were exposed to 0% O2 for 18 h, followed by immunoblotting for STAT6 and p-AKT (b), and quantification of BNIP3, PML, and REDD1 mRNA by RT-qPCR (c). d Immunoblot of p-TSC2, TSC2, and STAT6 in cells exposed to 0% O2 for the indicated periods (left) and summary data (right) showing p-TSC2 levels, expressed as fold changes relative to that in CONsi-transfected cells exposed to 21% O2. e Immunoblot of Rheb in mTOR immunoprecipitates (IPs) from U87MG cells transfected with CONsi or STAT6si and exposed to 21% or 0% O2 for 10 h. f Immunoblot of STAT6 in mTOR IPs from U87MG cells exposed to 0% O2 for 6 h. g Immunoblot of Rheb in STAT6 IPs from U87MG cells exposed to 0% O2 for 10 h. IgG IPs were used as a negative control. h Immunoblot of Rheb in STAT6 IPs from U87MG cells exposed to 0% O2 for the indicated periods. i Immunoblot of Rheb in STAT6 IPs from U87MG cells exposed 0% O2 for 3 h (H3h), followed by reoxygenation for 12 h (H3h/R). j Top, schematic of STAT6 deletion mutants. α-H, a-helicase domain; DBD, DNA-binding domain; LD, linker domain; SH2: src homology 2 domain; TAD, transactivation domain. FL, full length; d1-d4, deletion of residues 1–260 (d1), 261–430 (d2), 431–522(d3), or 523–622 (d4), respectively. Bottom, immunoblot of Rheb in Myc-tagged STAT6 (Myc-STAT6) IPs from U373MG cells transfected with the indicated Myc-STAT6 deletions and exposed to 0% O2 for 6 h