Volume 118, Issue 5, Pages 859-866 (May 2000) Expression of glucocorticoid receptor β in lymphocytes of patients with glucocorticoid- resistant ulcerative colitis  Mitsunori Honda, Fumika Orii, Tokiyoshi Ayabe, Shinjiro Imai, Toshifumi Ashida, Takeshi Obara, Yutaka Kohgo  Gastroenterology  Volume 118, Issue 5, Pages 859-866 (May 2000) DOI: 10.1016/S0016-5085(00)70172-7 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of GR mRNAs and GR proteins in PBMCs of patients with UC. Total RNA extracted from PBMCs of cases 2, 5, 10, 14, and 19 was reverse-transcribed using random 9mer primers. The resulting cDNA was amplified using either (B) hGRα-specific primers or (C) hGRβ-specific primers. (A) To serve as a quality control of cDNA samples, human GPDH was amplified from the same cDNAs. Resulting PCR products were separated by 1.5% agarose gel electrophoresis and stained by ethidium bromide. The sizes of the DNA markers (MW) are 603, 310, and 281 bp. Cell lysates of PBMCs from same patients were immunoprecipitated and enriched with anti–N terminus of hGR antibody and protein G–Sepharose. Resulting immunoprecipitate was subjected to SDS-PAGE and Western blotting. (D) Ninety-four–kilodalton hGRα-specific bands detected with hGRα-specific antibody in all samples of UC patients. (E) Ninety-kilodalton hGRβ-specific bands detected with hGRβ-specific antibody in samples of cases 14 and 19, but not detected in samples of other 3 patients. Gastroenterology 2000 118, 859-866DOI: (10.1016/S0016-5085(00)70172-7) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Quantitative RT-PCR. (A) RNA from PBMCs of patient 13 was reverse-transcribed, and resulting cDNA was amplified using hGRα- or hGRβ-specific primers. Aliquots of the PCR reaction were removed at 2-cycle intervals and electrophoresed. (B) After staining with ethidium bromide, fluorescence intensity of each lane was measured and plotted to generate amplification curves for both hGRα and hGRβ fragments. Forty-six cycles was required to amplify the same amount of hGRβ PCR product as hGRα PCR product on 36 cycles. (C) Template cDNA was sequentially diluted (for hGRα: 10, 20, 40, 80, 160, 320, and 640; for hGRβ: 2, 4, and 8) to analyze the hGRα/hGRβ mRNA ratio. Resulting PCR products at 40 cycles of each reaction were electrophoresed and stained by ethidium bromide. (D) Fluorescence intensity of each PCR product was measured and plotted as a function of dilution fold. The hGRα/hGRβ mRNA ratio was determined as 605-fold by this plot. Gastroenterology 2000 118, 859-866DOI: (10.1016/S0016-5085(00)70172-7) Copyright © 2000 American Gastroenterological Association Terms and Conditions