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. 2016 Aug 31;7:12696. doi: 10.1038/ncomms12696

Figure 2. Insulin promotes Akt-mediated phosphorylation of Bmal1 at Ser42.

Figure 2

Immunoblotting analysis of proteins recognized by phospho-Ser/Thr-Akt substrate antiserum in FLAG immunoprecipitates (IP) prepared from (a) primary hepatocytes infected with Ad-FLAG-Bmal1 or Ad-GFP, (b) primary WT or Akt2 null (Akt2 KO) hepatocytes infected with Ad-FLAG-Bmal1 in the presence or absence of insulin (INS, 50 nM, 30 min). (c) Immunoblotting analysis of endogenous Bmal1 proteins co-immunoprecipitated by anti-FLAG antibody from lysates of HEK 293T cells transfected with FLAG-Akt2-CA (top), the corresponding gel was silver-stained (bottom). (d) Immunoblotting analysis of FLAG-Bmal1 proteins recognized by FLAG-HRP antiserum in phospho-Ser/Thr-Akt substrate immunoprecipitates prepared from HEK 293T cells transfected with indicated plasmids. (e) Immunoblotting analysis of pSer42-Bmal1 protein amounts in liver homogenates from mice fasted from ZT0, injected intraperitoneally with insulin (INS, 2 U kg−1) at ZT6 and then killed at ZT7. (f) Immunoblotting analysis of pSer42-Bmal1 protein amounts in primary WT or Akt2 null (Akt2 KO) hepatocytes exposed to insulin (INS, 50 nM) for indicated times. (g) In vitro kinase assay of Bmal1 protein phosphorylated by active recombinant Akt in immunoprecipitates of WT or Ser42Ala mutant (S42A) FLAG-Bmal1 from HEK 293T cell lysates. Ser42-phosphorylated Bmal1 (pSer42-Bmal1) was detected by phospho (Ser42) specific Bmal1 antiserum, the corresponding gel was Coomassie Brilliant Blue-stained (CBB, bottom). (h) Immunoblotting analysis of Ser42-phosphorylated Bmal1 (pSer42-Bmal1) protein amounts in Ad-GFP, Ad-FLAG-tagged WT or S42A mutant Bmal1 virus-infected primary hepatocytes treated with insulin (50 nM, 30 min) in the presence or absence of rapamycin (100 nM, 1 h pretreatment).