1. The Melanocortin 5 Receptor is Expressed in Human Sebaceous Glands and Rat Preputial Cells 
Diane Thiboutot, Aruntha Sivarajah, Kathryn Gilliland, Zhaoyuan Cong, Gary Clawson 
Journal of Investigative Dermatology 
Volume 115, Issue 4, Pages (October 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Amplified sequence consistent with the MC5-R is detected in human SGs by RT-PCR. Total RNA was extracted from microdissected human SG and treated with DNase. cDNA was synthesized and PCR was performed for each of the five melanocortin receptor subtypes and positive and negative controls. The gel was stained with ethidium bromide. Lane M, 100 bp markers; lane 1, positive control (PCR products from the RT-PCR reaction performed on the positive control RNA supplied in the kit); lane 2, positive control (PCR products using primers to the human β-actin gene); lane 3, MC1-R; lane 4, MC2-R; lane 5, MC3-R; lane 6, MC4-R; lane 7, MC5-R; lane 8, negative control (no reverse transcriptase; SG RNA, reacted with β-actin primers). Expected sizes of amplified sequences based on primer design were 500 bp for the kit positive RNA control; 250 bp for the β-actin positive control; 733 bp for MC1-R, 304 bp for MC2-R, 178 bp for MC3-R, 568 bp for MC4-R, and 460 bp for MC5-R. Bands consistent with positive controls, MC4-R, and MC5-R are detected. Sequence verification was obained for only the MC5-R.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
MC5-R antibody specificity is confirmed in blocking experiments. Western blotting of a rat preputial gland extract was performed. A 1:10 dilution of IgY was preincubated in the presence and absence of the MC5-R peptide. Lane M, molecular weight markers; lane A, test IgY alone; lane B, test IgY preincubated with MC5-R peptide. The 43 kDa band representing the MC5-R was diminished by preincubation of the antibodies with the peptide, confirming specificity of the generated antibodies.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
MC5-R is detected in human facial skin and sebocytes. Immunohistochemistry was performed on cryostat sections of human facial skin and human sebocytes using a 1:25 dilution of affinity-purified IgY. (A) Negative control, human facial skin incubated in the absence of primary antibody. (B) Human facial skin: MC5-R is detected in epidermis (large arrow), hair follicles, SGs, eccrine glands, and endothelial cells (small arrow). (C) Human facial skin: high power view reveals localization of MC5-R in hair follicles (large arrow), sebaceous duct (small arrow), and basal layer of the SG. (D) Negative control, human facial sebocytes incubated in the absence of primary antibody. (E) Human facial sebocytes: MC5-R is detected in sebocytes (arrow) and surrounding 3T3 fibroblasts. (F) Human facial sebocytes: high power view reveals cytoplasmic localization of MC5-R in sebocytes (arrow). Scale bars: 75 μm [scale bar in part (B) refers to parts (A), (B), and (E); scale bar in part (C) refers to parts (C), (D), and (F)].
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
MC5-R is detected in rat skin and rat preputial sebocytes. Immunohistochemistry was performed on cryostat sections of rat skin and rat preputial sebocytes using a 1:25 dilution of the affinity-purified IgY. (A) Negative control, rat skin incubated in the absence of primary antibody. (B) Rat skin: MC5-R is detected in epidermis (large arrow), hair follicles (small arrow), SGs, eccrine glands, and endothelial cells. (C) Negative control, rat preputial sebocytes (arrow) incubated in the absence of primary antibody. (D) Rat preputial sebocytes: cytoplasmic distribution of MC5-R is detected. Scale bars: 75 μm [scale bar in part (B) refers to parts (A), (B), and (C)].
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
MC5-R protein is detected in Western blots of human SG and rat preputial gland extracts. Blots were incubated in the presence of a 1:10 dilution of IgY. Lane M, molecular weight markers; lane A, rat preputial gland extract; lane B, human SG extract. 43 kDa bands consistent with the MC5-R antibody were detected in both rat preputial glands and human SGs, confirming the presence of MC5-R protein in each tissue.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions