Figure 4. RT4-D6P2T characterization.
(A) Western analysis revealed the RT4 Schwannoma cell line, as opposed to NIH3T3 fibroblast line, expresses the higher molecular weight secreted periostin isoform, similar to the multiple isoforms expressed endogenously in vivo in newborn mice pups. While RT4 cells predominantly express two isoforms (~90kDa & ~82kDa) and RT4 supernanatant contains secreted (~90kDa) periostin, the NIH3T3 cells only express the lower molecular weight (~82kDa) isoform within the cells and do not secrete periostin. Note that there is also a complete absence of periostin in the newborn nulls, verifying antibody specificity. Equal loading was ensured via normalizing for actin levels and loading equivalent amounts of supernatant (verified via Coomassie blue staining, not shown). (B–D) RT4 cells were grown on uncoated glass culture slides and briefly fixed with 4% PFA prior to immunostaining. (B) Anti S-100 staining, with hematoxylin nuclear counterstaining. Note cytoplasmic S-100 expression and absence of specific signal in negative control which did not receive the primary antibody (inset in B). (C) Anti-Sox10 immunofluorescent staining. Note appropriately localized Sox10 expression in nucleus and same view as phase-contrast (inset in C). (D) Anti-periostin staining, with methyl green nuclear counterstain and DIC optics. Note expression of periostin localized to the cytoplasm and that no expression is evident in negative control stained with pre-immune in place of primary periostin antibody (inset in D). The absence of staining in negative (−ve) control samples demonstrated that staining was specific. Magnification: all images 400X.