COS-1 cells transiently transfected with either HA hgr wt, HA hgr S211A or HA hgr S226A

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1 1 SUPPLEMENTRY FIGURES Fig. 1 & Specificity of the nti-p-s211 nd nti-p-s226 ntibodies COS-1 cells trnsiently trnsfected with either H hgr wt, H hgr S211 or H hgr S226 were treted with 1nM Dex for 1 hour. Western blot nlysis ws performed using either n nti-p-s211 GR-specific ntibody () or nti-p-s226 GR-specific ntibody (). Therefter the membrne ws stripped nd reprobed with nti-gr ntibody (totl GR). Dex WT S226 Wt S211 Dex Dex P-S211 P-S226

2 2 Fig. 11 -D Determining the optiml concentrtion of α-mnitin nd MG132 COS-1 cells trnsiently trnsfected with ptt-gre, pβgl nd ph-hgr were pre-treted with α-mnitin for 24 hours before tretment with vehicle () or 1 nm dex for n dditionl 24 hours. Cells were hrvested nd either luciferse (luc) ctivity ws mesured nd normlised to totl protein per well s determined by rdford ssys (), or β glctosidse (βgl) ctivity ws determined (). To block protesoml GR degrdtion, COS-1 cells trnsiently trnsfected with ph-hgr were pretreted with MG132 for 4 hours before tretment with vehicle () or 1 nm dex for n dditionl 24 hours. Equl mounts of protein were seprted on SDS-PGE nd Western blotting nlysis ws performed probing for GR nd β-ctin (C). fter quntifiction nd normliztion, the GR levels in the presence of ethnol vehicle () but in the bsence of MG132, were set to 1% nd the GR levels in the other smples were clculted s percentge thereof (D). 15 Luc 2 β-gl Rel. Trnsctivtion 1 5 b c,c Dex Dex Dex µg/ml 1µg/ml 2.5µg/ml α-mnitin Rel luc Dex Dex Dex µg/ml 1µg/ml 2.5µg/ml α-mnitin

3 3 C MG132 µm 5µM 1µM Dex Dex Dex Dex Dex Dex β-ctin D mount of GR b Dex Dex Dex µm 5µM 1µM MG132

4 4 Fig. 12 & Muttion of S23, S211 nd S226 COS-1 cells trnsiently trnsfected with either prs hgr wt or prs hgr S23/211/226 were treted with 1nM Dex for 1 hour. Western lot nlysis ws performed using either n nti-p- S211 GR-specific ntibody () or n nti-p-s226 GR-specific ntibody (). Therefter the membrne ws stripped nd reprobed with nti-gr ntibody. Wt Dex S23/211/226 Dex P-S211 GR

5 5 Fig Reltive mount of GR nd GRIP-1 immunoprecipitted COS-1 trnsiently trnsfected with ph-grip-1, s well s either prs-hgr (Wt), prs-3 (3) or empty vector (-GR) were treted with vehicle () or 1 nm dex for 1 h. fter lysis, 1/5 of the cell lyste ws subjected to SDS-PGE nd Western blotting nd nlysed for input levels of GR nd H-GRIP-1. The reminder of the cell lyste ws immunoprecipitted using nti-gr ntibody nd protein /G beds. Proteins bound to the complex were nlysed by Western blotting nd probing for GR nd H-GRIP-1. The mounts of GR () nd H-GRIP-1 () immunoprecipitted were quntified nd plotted s percentge of their reltive inputs. Results show the verge ± SEM of between two to four independent experiments. The letters nd b re used to denote sttisticlly significnt differences, such tht ll the conditions with the sme letter re not sttisticlly significntly different from ech other (P>.5), while those hving different letters re sttisticlly significntly different from ech other (P<.5). Sttisticl significnce of differences is denoted by *** to indicte P<.1. Rel. GR immunoprecipitted (% Input; W t = 1) GR IP Wt Wt Dex 3 3 Dex -GR -GR Dex Rel. GRIP-1 immunoprecipitted (% Input; Wt = 1) GRIP IP b Wt Wt Dex 3 3 Dex -GR -GR Dex

6 6 Fig Reltive mount of GR nd GRIP-1 bound to the GRE-beds COS-7 cytosols (2 µl) contining expressed prs-hgr (Wt), prs-3 (3) or empty vector (- GR) were incubted with vehicle () or 1 µm dex. Therefter, the cytosols were incubted with biotinylted GRE oligonucleotides ttched to Streptvidin beds followed by incubtion with COS-7 cytosols contining overexpressed H-GRIP-1. Equl mounts of input cytosol, s well s DN-bound proteins were seprted by SDS-PGE nd visulized by Western blotting probing with nti-h nd nti-gr ntibodies. fter quntifiction the mounts of GR () nd H-GRIP-1 immunoprecipitted with the GRE-beds, were clculted s percentge of their respective inputs. The grph shows pooled results of three independent experiments In () sttisticl significnce of differences is denoted by *** to indicte P<.1. Rel. GR bound to GREs (Wt = 1) GR Wt Wt Dex 3 3 Dex -GR -GR Dex Rel. GRIP-1 recruited to GREs (Wt = 1) *** GRIP-1 Wt Wt Dex 3 3 Dex -GR -GR Dex