Figure 5.

Cloning, Western blot, and indirect immunofluorescence analyses of mGRβ. A, Primers spanning the ATG start site in exon 2 and the distal region of intron 8 (see Fig. 2) were used to isolate the full-length mGRβ cDNA, followed by cloning into pcDNA3.1 to yield the pMGRβ-H57 vector. COS-7 cells were transfected with 8 μg each of pSV2Wrec (mGRα), pMGRβ-H57 (mGRβ), or empty vector (mock), followed by Western blot analysis with FiGR mouse monoclonal antibody that recognizes a common epitope on both mGR isoforms. B, A rabbit polyclonal antibody specific to mGRβ (rMGRβ-Ab) was generated using the unique 15-amino acid terminal sequence. Whole-cell extracts from COS-7 cells transfected with pSV2Wrec, pMGRβ-H57, or empty vector (mock) were simultaneously probed with FiGR and rMGRβ-Ab antibodies. The Odyssey infrared detection system utilizing 680 nm (red) and 800 nm (green) emitting counter antibodies was used to detect FiGR (mGRα) and rMGRβ-Ab, respectively. Results show that the rMGRβ-Ab antibody reacts only with mGRβ not with mGRα. C, MEF cell lysates were immunoadsorbed with rMGRβ-Ab (I) or preimmune (PI) serum, followed by blotting with rMGRβ-Ab, using enhanced chemiluminescence. Whole-cell extracts from COS-7 cells transfected with pMGRβ-H57 were used for comparison. D, Two separate MEF cell lysates were immunoadsorbed with FiGR (I) or nonimmune (NI) IgG, followed by sequential blotting with rMGRβ-Ab and FiGR, using enhanced chemiluminescence. Relative densitometric ratios of mGRβ to mGRα are shown. E, Indirect immunofluorescence using preimmune serum, rMGRβ-Ab, or rMGRβ-Ab blocked with mGRβ peptide was performed on MEF cells treated or untreated with Dex (100 nm, 2 h).