Detection of MuSK expression in HEp-2 M4 cells.

<p>A. Exponentially growing transfected clone HEp-2 M4 and non-transfected parental HEp-2 cells were used for RNA preparation and RT-PCR analysis of heterologously expressed MuSK, with (+) or without (−) reverse transcriptase (RT), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083924#s2" target="_blank">materials and methods</a>. 1, HEp-2 M4+RT; 2, HEp-2 M4−RT; 3, HEp-2+RT; 4, HEp-2−RT. Amplicons were analyzed by agarose electrophoresis with 50 bp DNA size marker as control (5). B. HEp-2 M4 cells growing on glass slides were fixed and processed for indirect immunofluorescence with anti-V5 antibody to detect expression of the MuSK-V5 fusion protein as described in the methods section. Photo was taken by AKLIDES system (10× objective, TRANSFECT mode). C. Living HEp-2 M4 cells grown on glass slides were incubated with commercial anti-MuSK primary antibody followed by fixation and secondary antibody staining as described in the methods section. The image was taken by an Olympus fluorescence microscope IX81 (40× objective). MuSK-specific signals appear as characteristic speckled pattern. D. Living HEp-2 M4 cells grown on glass slides were incubated with anti-MuSK autoantibody positive patient sera (1) and further processed for double immunofluorescence staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083924#s2" target="_blank">materials and methods</a>. A second serum pretested by RIA to be negative for anti-MuSK autoantibody (2) was used as negative control. Detection of the V5 epitope by V5-specific primary antibody and Cy3 labeled secondary antibodies, and simultaneous detection of MuSK with patient autoantibodies and Alexa Fluor 488 conjugated secondary antibodies is illustrated as indicated with V5 (Cy3) and patient serum (FITC channel used), respectively. Double immunofluorescence staining images were obtained by merging both images as indicated (merge). The double immunofluorescence staining analysis shows strong evidence for specific binding of MuSK autoantibodies to only those HEp-2 M4 cells expressing the MuSK-V5 fusion protein. Images were taken by the Olympus fluorescence microscope IX81 (40× objective). Scale bar 50 µm.