Fig 2: Binding of CRTC2 to Cebpd-associated enhancer is hindered by JAK inhibition. (a–c) ChIP-Seq of CRTC2 from 3T3-L1 cells treated with either vehicle or P6 was stimulated with forskolin. A total of 17 552 CTRC2 peaks were identified in forskolin-treated cells. (a) MA plot showing CRTC binding affected by P6. Differentially bound CRTC2 was identified using EdgeR and the false discovery rate (FDR) of individual CTRC2-binding sites is indicated in the plot. (b) Boxplots of CREB, AP-1 and STAT motif strength within CRTC2-binding sites either unaffected or reduced by P6. Unchanged binding is defined as differential binding with FDR>0.8 and reduced binding is defined as differential binding with FDR<0.2 and log2 fold change <0. (c) ChIP-seq profiles of Cebpd locus. STAT1, STAT3 and DHS data are from Siersbæk et al. (REF). Primer sets used for 3C analysis are indicated together with HindIII sites. Primers 1 and 2 anneal at the HindIII sites surrounding the Cebpd TSS. Primers 3 and 4 anneal at a DNase inaccessible control region 40 kb downstream of the TSS and primers 5 and 6 anneal at the 61 kb enhancer occupied by CRTC2 and STATs. (d) 3C analysis of interaction between 61 kb enhancer and control region with Cebpd TSS. Ligation efficiency was normalized to ligation efficiency of HindIII-digested BAC RP23-77N6 containing the Cebpd locus

Fig 3: CRTCs can bind STAT3, STAT5 and MDM2. (a) 293T cells were transfected with FLAG-tagged CRTCs and/or STATs. FLAG-immunoprecipitates (IPs) were analyzed for STAT3 and STAT5 by western blotting. (b) 293T cells were transfected with MYC-tagged MDM2 and/or STATs. MYC-IPs were analyzed for STAT3 and STAT5 by western blotting. (c) 293T cells were transfected with MYC-tagged MDM2 and/or JAK1. MYC-IPs were analyzed for JAK1 by western blotting. (d) 293T cells were transfected with FLAG-tagged CRTC2 and/or MDM2. FLAG-IPs were analyzed for MDM2 by western blotting. (e) 293T cells were transfected with FLAG-tagged CRTC3 and/or MDM2. FLAG-IPs were analyzed for MDM2 by western blotting