Fig. 1
Assembly and expression of ER IBs recognizing ST8SiaII and ST8SiaIV. a PCR amplification of the variable domains VH and VL of anti-ST8SiaII and anti-ST8SiaiIV scFv fragments (left side) and assembly of the corresponding scFvs (right side). 20 μl of the PCR products were analyzed on a 1% agarose gel (M = 100 bp ladder). b Primary sequence of anti-ST8SiaII and anti-ST8SiaIV intrabody. Shown are the coding (lower lane) and amino acid sequence (upper lane) of anti-ST8SiaII-IB and anti-ST8SiaIV-IB including the ER signal peptide, the myc epitope and the ER retention sequence. The complementary-determing regions (CDR1-CDR3) of the variable domains of the heavy and light chain are printed in blue letters. The synthetic linker, shown in red letters, localized between the VH and VL domains was introduced by assembly PCR. c Immunofluorescence analysis by microscopy of permeabilized and fixed HEK293 cells transiently transfected with IBs. Intracellular expression was stained in red. Positive control: anti-TLR9 IB, negative control: HEK 293 cells transiently transfected with the empty expression plasmid pCMV/myc/ER. d Immunoblot analysis, Expression of anti-ST8SiaII-IB and anti-ST8SiaIV-IB visualized with peroxidase labelled secondary antibody. Sample volume: 10 μl of 100 μl cell lysat from 106 cells transiently transfected for 48 h with the intrabody DNA in a 6-well microtiter plate