Supplementary Materialsjcm-08-01863-s001. can help to form subgroups of SS for targeted therapy. = 35) and systemic lupus erythematosus (SLE, = 35) individuals without signs and symptoms suggestive of SS were from the Division of Rheumatology, Seoul St. Marys Hospital. RA and SLE were chosen as disease settings because they are most prevalent other than SS among the connective cells diseases, particularly in Korea [14,15]. The demographic and laboratory characteristics of PKI 14-22 amide, myristoylated the subject groups are summarized in Table 1. Accompanying clinical and laboratory data were also obtained from the serum providers. Due to the shortage in the available amount of sera, SLE and RA samples were subjected only to screening of anti-AQP5 IgG by ELISA. Table 1 Demographic and laboratory characteristics of the subjects. = 111)= 43)= 35)= 35)(%)102 (91.9)0 (0)20 (69.0), 29?NDanti-SSB+, (%)61 (55.0)0 (0)6 (20.7), 29?NDRF+, (%)75 (67.6)0 (0)0 PKI 14-22 amide, myristoylated (0), 22?27 (77.1)ANA+, (%)75 (67.6)0 (0)34 (97.1)14 (45.2), 31?UWSFR 0.1 mL/min, (%)84 (75.7)15 (34.9)NDNDLabial salivary gland biopsy resultsFLS score 1, (%)80 (80), 100?0 (0)NDNDFLS score 0 << 1, (%)7 (7), 100?8 (18.6)NDNDFLS score = 0, (%)13 (13), 100?35 (81.4)NDNDN/SCS13 (13), 100?34 (79.0)NDNDSchirmers test 5 mm in 5 min, (%)63 (57.3), 110?11 (25.6)NDNDOcular staining score 3, (%)105 (95.5), 110?0 (0)NDND Open in a separate window ND: not done; SSA: Sj?grens syndrome-related antigen A; SSB: Sj?grens syndrome-related antigen B; RF: rheumatoid factor; ANA: antinuclear antibody; UWSFR: unstimulated whole salivary flow rate; FLS: focal lymphocytic sialadenitis; N/SCS: nonspecific/sclerosing chronic sialadenitis. *Significantly different from the other groups by ANOVA with Bonferroni-adjusted post hoc tests; ? presents the total number with proper data. 2.2. Cell-Based Immunofluorescence Cytochemistry (CB-IFC) Madin-Darby canine kidney (MDCK) cells expressing full length human SHC2 AQP5 (MDCK-AQP5) were cultured in DMEM with 10% FBS and 1% penicillin and streptomycin in the presence of 2 mg/ml G418 [16]. A mixture of MDCK and MDCK-AQP5 at 1:1 was plated onto collagen-coated coverslips 12 mm in diameter. The cells were stimulated with 0.5 mM cAMP for 24 h, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), and then subjected to PKI 14-22 amide, myristoylated antigen retrieval by incubation in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH = 6) at 105 C for 20 min. After blocking with 5% bovine serum albumin (BSA) in PBS, the cells were incubated with mouse monoclonal antibodies (a clone D-7) raised against a peptide near the C-terminus of human AQP5 (Santa Cruz Biotechnology, Dallas, TX, USA), along with human serum (1:10 for IgA and 1:100 dilutions for IgG). In the case of anti-AQP5 IgG screening, cells were incubated in parallel with sera preincubated overnight with 10 g/ml synthetic peptide A, C2, or E1 in RIA buffer (10 mM Tris-HCl, 350 mM NaCl, 1% BSA, 1% Triton X-100, 10% horse serum, pH = 7.6). The sequences of peptides have been previously reported [12]. Subsequently, the cells were stained with Alexa Fluor 488Cconjugated rat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) and either Alexa Fluor 555Cconjugated goat anti-human IgG (Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 594Cconjugated rabbit anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA). After mounting, the cells had been analyzed by confocal microscopy (Carl Zeiss, Oberkochen, Germany). At least 3 regions of AQP5-expressing cells had been randomly selected predicated on staining from the mouse anti-AQP5 antibody and imaged sequentially for staining by either human being IgA or IgG. After coding the pictures, the relative intensities from the anti-AQP5 human Ig signals were dependant on blindly.
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