Flower protein is expressed in primary CTLs from mouse. (A) Western blot of lysates from whole-brain, stimulated (stim) CTLs and naive CTLs prepared from WT or Flower (mFl)-deficient mice (KO) by using the anti-Flower antibody and, to control protein loading, anti-GAPDH antibody. 20 µg total lysate was loaded per lane. The Flower protein is detected only in WT lysates at the expected molecular mass of ∼18 kD. (B) Generation of the mFl^−/− mouse (see Fig. S2 for details). Scheme of the nontranslated (open boxes) and translated exons (closed boxes; not in scale, taken from Ensembl Genome Browser). WT mFl allele, targeting construct (HTGRS6009_A_G03; Eucomm), and recombinant mFl^L3F2, mFl^L2F1, and mFl KO alleles are shown. In the mFl^L3F2 allele, exons 2 and 3 are flanked by lox P sites (closed triangles). An FRT (open triangles) sequence-flanked gene cassette comprises the SA-IRES-βGal followed by a promoter-driven neo cassette. Flp recombinase-mediated conversion of the L3F2 allele to the L2F1 allele and Cre recombinase-mediated conversion of the L2F1 to the KO allele (L1F1) are shown. DTA, diphtheria toxin A. (C) Cartoon showing the topology of Flower. The green circles indicate the position of the pHluorin fluorophore. N, N terminus; C, C terminus. (D) Localization of endogenous (top) and overexpressed (bottom) Flower-HA protein by using anti-Flower antibody in WT and Flower KO CTLs. Bars, 5 µm.