Confirmation of Crry gene mutation at mRNA and protein levels. (A) RT-PCR analysis confirming thymus-specific production of a shortened Crry mRNA in CD4-Cre⁺ and Lck-Cre⁺-Crry^flox/flox mice. LS indicates leader sequence; SCR, short consensus repeat; UTR, untranslated region; TM, transmembrane domain; Kid, kidney; Thy, thymus. (B) Domain structures of the WT (full-length, containing SCR 1-5) and mutant Crry protein (CrryΔ3-4, containing SCR 1, 2, and 5) predicted from the shortened Crry mRNA. (C) FACS analysis of CHO cells stably transfected with the full-length Crry cDNA or the shortened Crry cDNA (CrryΔ3-4). Both proteins were expressed and detected on the cell surface by a polyclonal rabbit anti–mouse Crry antibody (α-Crry). Cells transfected with the empty pCDNA3 vector were used as controls (CHO). (D) Western blot analysis of CHO cells stably transfected with the full-length Crry (lane 1), CrryΔ3-4 (lane 2), or pCDNA vector (lane 3). As expected, CrryΔ3-4 produced a protein of smaller size. A polyclonal rabbit anti–mouse Crry antibody (α-Crry) was used. (E) Left: Western blot using α-Crry analysis indicating thymus-specific production of the mutant Crry protein (smaller size between 36.9 kDa and 50.2 kDa). Right: Absorbed SCR3/4-specific anti-Crry antibody detected WT Crry protein but not the mutant protein in the thymus. (F) Confirmation of the specificity of α-SCR3/4 by FACS analysis of transfected CHO cells. It detected full-length Crry but not CrryΔ3-4 expressed on CHO cells.