D 
a mouse secondary antibody, sheep anti-mouse IgGHRP (GE Healthcare, NXA931) at a dilution of 1:10.000.Statistical AnalysisData is presented in mean6S.D. The Student’s t test (tail 2, type 2) was used for statistical analysis and differences were considered significant when p-value was ,0.01.Supporting InformationFigure S1 Characterization of skeletal muscle specific IGF-1 transgenic mouse lines. (A) Transgene expression in founder MLC/IGF-1 lines assessed by Northern blot analysis on 10 ug of total RNA of each tissue with a probe specific for the SV40 polyadenylation sequence. Transgenic samples of 22948146 every line were compared to WT littermates. (B) Comparison of transgene expression levels between the four MLC/IGF-1 lines by Northern blot analysis on 10 ug of total RNA from quadriceps muscle using a SV40-specific probe. (TIF) Figure SImmunohistochemistryIGF-1 concentrations in media from HEK 293 cells transfected with IGF-1 encoding constructs were determined and the amount of IGF-1 was adjusted (using media from untransfected cells) to 200 ng/mL. Sections of decellularized tissue were deparaffinised using xylene and rehydrated by passaging through solutions of decreasing ethanol concentrations, then incubated in the normalized IGF-1 conditioned media. After removal of the media, the sections were washed and fixed in 4 formaldehyde, blocked in 5 normal donkey serum, and incubated with the primary antibody (goat anti-mouse-IGF-1 (Sigma, I-8773) at 1:6 dilution in blocking buffer at 4C overnight). Subsequently, the sections were incubated with biotinylated horse anti-goat IgG antibody (Vector Labs, BA-9500) at 1:200 dilution at 4C for 48?2 hrs followed by incubation with streptavidin-Alexa 594 (Invitrogen, S-11227) forDeglycosylation of IGF-1 propeptides increase their affinity to negatively charged surfaces. A) Growth medium from transiently transfected HEK 293 cells incubated with (lanes 2 and 4) and without (lanes 1 and 3) deglycosylation enzyme mix (IGF-1 levels normalised to 200 ng/ mL; 20ml load). B) Binding of deglycosylated (lanes 6 and 8) and non-deglycosylated (lanes 5 and 7) IGF-1 propeptides to negatively charged tissue culture plates. (TIF)AcknowledgmentsWe thank Esfir Slonimsky for excellent Docosahexaenoyl ethanolamide technical assistance, and members of the Rosenthal laboratory for critical comments and helpful discussions.Author ContributionsConceived and designed the experiments: MSH ES. Performed the experiments: MSH ES AP EP NW TN. Analyzed the data: MSH ES AP NW. Wrote the paper: MSH ES NR.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread environmental contaminants that produce a variety of toxic and biological effects, most of which are mediated by the aryl hydrocarbon (dioxin) receptor (AhR), a ligand-dependent nuclear receptor [1?]. 23388095 Although most TCDD-like AhR agonists are 56-59-7 site structurally related, recent studies suggest a high degree of promiscuity in the ligand binding specificity of the AhR [6?]. During our analysis of solvent extracts of food products for AhR agonists [10], the inadvertent use of a rubber cap liner instead of a Teflon cap liner on vials containing the extracting solvent (DMSO) revealed that chemicals readily extracted from the rubber cap liner could maximally activate AhR DNA binding; no activation was observed with DMSO stored in Teflon-capped vials. These results, coupled with our recent identification of AhR agonists in extracts of commercial newspapers [11,12] and automob.D a mouse secondary antibody, sheep anti-mouse IgGHRP (GE Healthcare, NXA931) at a dilution of 1:10.000.Statistical AnalysisData is presented in mean6S.D. The Student’s t test (tail 2, type 2) was used for statistical analysis and differences were considered significant when p-value was ,0.01.Supporting InformationFigure S1 Characterization of skeletal muscle specific IGF-1 transgenic mouse lines. (A) Transgene expression in founder MLC/IGF-1 lines assessed by Northern blot analysis on 10 ug of total RNA of each tissue with a probe specific for the SV40 polyadenylation sequence. Transgenic samples of 22948146 every line were compared to WT littermates. (B) Comparison of transgene expression levels between the four MLC/IGF-1 lines by Northern blot analysis on 10 ug of total RNA from quadriceps muscle using a SV40-specific probe. (TIF) Figure SImmunohistochemistryIGF-1 concentrations in media from HEK 293 cells transfected with IGF-1 encoding constructs were determined and the amount of IGF-1 was adjusted (using media from untransfected cells) to 200 ng/mL. Sections of decellularized tissue were deparaffinised using xylene and rehydrated by passaging through solutions of decreasing ethanol concentrations, then incubated in the normalized IGF-1 conditioned media. After removal of the media, the sections were washed and fixed in 4 formaldehyde, blocked in 5 normal donkey serum, and incubated with the primary antibody (goat anti-mouse-IGF-1 (Sigma, I-8773) at 1:6 dilution in blocking buffer at 4C overnight). Subsequently, the sections were incubated with biotinylated horse anti-goat IgG antibody (Vector Labs, BA-9500) at 1:200 dilution at 4C for 48?2 hrs followed 
by incubation with streptavidin-Alexa 594 (Invitrogen, S-11227) forDeglycosylation of IGF-1 propeptides increase their affinity to negatively charged surfaces. A) Growth medium from transiently transfected HEK 293 cells incubated with (lanes 2 and 4) and without (lanes 1 and 3) deglycosylation enzyme mix (IGF-1 levels normalised to 200 ng/ mL; 20ml load). B) Binding of deglycosylated (lanes 6 and 8) and non-deglycosylated (lanes 5 and 7) IGF-1 propeptides to negatively charged tissue culture plates. (TIF)AcknowledgmentsWe thank Esfir Slonimsky for excellent technical assistance, and members of the Rosenthal laboratory for critical comments and helpful discussions.Author ContributionsConceived and designed the experiments: MSH ES. Performed the experiments: MSH ES AP EP NW TN. Analyzed the data: MSH ES AP NW. Wrote the paper: MSH ES NR.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread environmental contaminants that produce a variety of toxic and biological effects, most of which are mediated by the aryl hydrocarbon (dioxin) receptor (AhR), a ligand-dependent nuclear receptor [1?]. 23388095 Although most TCDD-like AhR agonists are structurally related, recent studies suggest a high degree of promiscuity in the ligand binding specificity of the AhR [6?]. During our analysis of solvent extracts of food products for AhR agonists [10], the inadvertent use of a rubber cap liner instead of a Teflon cap liner on vials containing the extracting solvent (DMSO) revealed that chemicals readily extracted from the rubber cap liner could maximally activate AhR DNA binding; no activation was observed with DMSO stored in Teflon-capped vials. These results, coupled with our recent identification of AhR agonists in extracts of commercial newspapers [11,12] and automob.
