Specific binding of PRG3 antibodies to intra- and extracellular domains. (A) Schematic depiction of the amino acid sequence of rPRG3 with its predicted orientation in the plasma membrane. Transmembrane-spanning domains (grey) are labeled TM1-TM6 and the C- and N-terminal regions are located on the intracellular side. The putative N-glycosylation site at asparagine 163 is indicated in red, sites used to generate the PRG3 antibodies in blue, and loci that differ between PRG3 of mouse and rat in green. (B and C) Specificity analysis of the two anti-PRG3 antibodies revealed that anti-PRG3 53 (B) and anti-PRG3 296 (C) show similar immunoreactivity. HEK293 cell lysates overexpressing GFP-rPRG3 or endogenous PRG3 from mouse brain were probed with the antibodies under different conditions. Membranes probed with the native antibody (Western blot) showed a double band with either anti-PRG3 53 or anti-PRG3 296 (B and C, left). A two-hour preincubation of the antibody with BSA yielded the same result (B and C, middle), whereas a similar preincubation with the corresponding peptide abolished immunoreactivity (B and C, right). (D) Specificity analysis of the two anti-PRG3 antibodies revealed no cross reactivity to other PRG family members. Fusion proteins of several PRG family members with eGFP (PRG1, PRG2, PRG3, PRG4, and PRG5) were used in the Western blot analyses. Both used PRG3 antibodies (anti-PRG3 296 and 53) detected protein exclusively in the PRG3 lane as a double band.