Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal weight (A) and umbilical artery serum DHAS (B) and cortisol (C) levels in untreated baboons on days 100 (middle, n = 4) and 165 (late, n = 8) of gestation and on day 165 in animals that were treated daily on days 100–164 with letrozole (0.115 mg/kg body weight per day, n = 8) or letrozole plus estradiol (each at 0.115 mg/kg body weight per day, n = 5). Values indicated by different letter superscripts are different at P < .05 to P < .01 (ANOVA and Newman-Keul's multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques:

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Western immunoblot of MC2R (A) and MRAP (B) incubated with primary antibody (Ab), with primary Ab preabsorbed with blocking peptide or without primary Ab in the baboon fetal adrenal gland on day 165 of gestation in untreated and letrozole-treated baboons (n = 2 samples each). Panels C and D show Western immunoblots of MC2R and MRAP in the fetal liver and epididymis on day 165 in an untreated baboon.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Western Blot, Incubation, Blocking Assay

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MC2R protein expression assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole on days 100–164 (B and D). Panel E shows the means (±SE) of fetal adrenal MC2R protein expression, quantified by PLA and image analysis/Metamorph software, on day 100 (midgestation, n = 4) and day 165 (late gestation, n = 8) in untreated baboons and on day 165 in animals treated with letrozole (n = 8) or letrozole plus estradiol (n = 5). Each red PLA signal represents a single molecule of MC2R protein detected by primary MC2R antibody tagged with a secondary antibody conjugated to fluorescently labeled oligonucleotide. Nuclei are labeled blue. P-450C17 immunostaining with Alexa Fluor 488-conjugated IgG within TZ (A and B) and FZ (C and D) cells is shown in green. Final magnification, ×400 (A–D).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, Software, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MRAP protein expression assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole (B and D). Panel E shows the means (±SE) of fetal adrenal MRAP quantified by PLA and image analysis/Metamorph software in the same baboons in which MC2R is shown in Figure 3. Nuclei are labeled blue. P-450C17 immunostaining within TZ (A and B) and FZ (C and D) are shown in green. Final magnification, ×400 (A–D). *, Significantly different at P < .03 in late vs midgestation.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, Software, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MC2R-MRAP protein interaction assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole (B and D). Panel E shows the means (±SE) of fetal adrenal MC2R-MRAP interaction quantified by PLA and image analysis/Metamorph software in the same baboons in which MC2R is shown in Figure 3. For PLA detection of MC2R-MRAP protein interaction, tissue was incubated with a secondary antirabbit PLUS antibody and a secondary antigoat minus antibody conjugated with oligonucleotide. Nuclei are labeled blue. P-450C17 immunostaining within TZ (A and B) and FZ (C and D) are shown in green. Final magnification, ×400 (A–D). *, Significantly different at P < .05 in letrozole-treated animals vs all other groups (ANOVA and Tukey-Kramer multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Software, Incubation, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal liver hepatocyte (A and B) and fetal epidydimal stroma (D and E) MC2R-MRAP interaction assessed by PLA on day 165 of gestation in untreated (A and D) and letrozole-treated (B and E) baboons. α-Smooth muscle actin immunostaining within fibromuscular cells of epidydimal ducts appears in green in panels D and E. Means ± SE of fetal liver (C) and fetal epidydimal stroma (F) MC2R-MRAP interaction were quantified by PLA in baboons untreated (n = 3) or treated with letrozole (n = 3). Final magnification, ×400 in each panel.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: A, Fetal pituitary POMC mRNA expression was quantified by in situ hybridization on days 100 (n = 3) and 165 (n = 7) in untreated baboons and on day 165 in animals treated with letrozole (n = 7) or letrozole plus estradiol (n = 7). B, Fetal (ie, umbilical artery) plasma ACTH levels on days 100 (n = 8) and 165 (n = 18) in untreated baboons and on day 165 in animals treated on days 100–164 with letrozole (n = 14) or letrozole plus estradiol (n = 11). Fetal pituitaries for POMC mRNA assay and blood samples for plasma ACTH assay were obtained from animals of the current study and from a contemporaneous group of baboons from our primate colony. Values indicated by different letter superscripts are different at P < .05 (ANOVA and Newman-Keul's multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, In Situ Hybridization

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Expression tests for ejection proteins gp14, gp15, and gp16 (A) Diagram of plasmids for gp14, gp15, and gp16. (B) Diagram of transformed colonies in different expression strains growing on agar plates treated with ampicillin (100 μg/mL). (C) Diagram of 100 mL starter cultures treated with ampicillin for different expression strains in 250 mL Erlenmeyer flasks. (D) Diagram of 1 L LB cultures treated with ampicillin and inoculated 1:50 with starter culture for different expression strains. (E–G) SDS-PAGE analysis for gp14, gp15, and gp16 before/after (−/+) induction with IPTG at OD600 = 0.6 and expression. Expected molecular weights are gp14, 21 kDa, gp15, 87 kDa and gp16, 146 kDa as incdicated on the SDS-PAGE gels in cyan, magenta, and green, respectively.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Expressing, Transformation Assay, SDS Page

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Workflow for the reconstitution of gp15:gp16 periplasmic tunnel of the T7 DNA-ejectosome.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques:

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Purification of T7 ejection proteins gp15 and gp16 (A) SDS-PAGE results for sonicated samples for gp15 and gp16. Abbreviations: M = Markers for molecular weight, T = Total sample post-sonication, S = Supernatant sample, and P = Pellet sample after centrifugation. (B) SDS-PAGE results for Ni-NTA purification for gp15 and gp16. Abbreviations: Ft = Flow-through sample that passed through the gravity-flow column without binding, W 1 and W 2 = Wash 1 and 2 eluent samples, E n = Elution samples, and B = Beads sample representing protein still bound to the Ni beads. (C) Chromatograms and SDS-PAGE results for gel filtration purified gp15 and gp16 on a calibrated analytical Superdex200 10/300 column. Only relevant lanes from the same gel are shown. Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Purification, SDS Page, Sonication, Molecular Weight, Centrifugation, Binding Assay, Filtration, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Purification of T7 ejection protein gp14 (A) SDS-PAGE results for sonicated, NLS detergent extracted, and Ni-NTA purified samples of gp14. Abbreviations: M = Markers for molecular weight, T = Total sample post-sonication, S = Supernatant sample, and P = Pellet sample after centrifugation, US = Ultracentrifuged Supernatant sample, UP = Ultracentrifuged Pellet sample, Ft = Flow-through sample that passed through the gravity-flow column without binding, W 1 and W 2 = Wash 1 and 2 eluent samples, E n = Elution samples, and B = Beads sample representing protein still bound to the Ni beads. (B) Chromatogram and SDS-PAGE results for gel filtration purified gp14 solubilized in NLS (extraction) detergent. (C) Chromatogram and SDS-PAGE results for gp14 detergent exchange from NLS to DDM detergent using an anion exchange column. (D) Chromatogram and SDS-PAGE results for gel filtration purified gp15 solubilized in DDM (mild) detergent. Only relevant lanes from the same gel are shown in (B–D). Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Purification, SDS Page, Sonication, Molecular Weight, Centrifugation, Binding Assay, Filtration, Extraction, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: In vitro assembly of the T7 periplasmic tunnel from purified gp15 and gp16 (A) Diagram of complex components incubated in a 3:1 molar ratio on ice for 60 min (B) Chromatogram and SDS-PAGE analysis of gel filtrated gp15:gp16 sample. Magenta arrow represents gp15 expected molecular weight at 87 kDa. Green arrow represents gp16 expected molecular weight at 146 kDa. Only relevant lanes from the same gel are shown. Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: In Vitro, Purification, Incubation, SDS Page, Molecular Weight, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Expected results for negative stain TEM and cryo-EM screening (A and B) Representative micrographs of (A) negatively stained and (B) vitrified gp15:gp16 complex. On the right-hand side are 2D-class averages. Scale bars, 100 nm. Reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Staining, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet:

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Virus, Recombinant, Electron Microscopy, Filtration, Plasmid Preparation, Software, Chromatography, Electrophoresis, Microscopy