Figure 1
Transcription and translation of short form of thymic stromal lymphopoietin (sfTSLP). (a) Relative mRNA expression of long form of TSLP (lfTSLP) and sfTSLP in oral mucosal biopsies (left, n=4) and cultured oral keratinocytes (KCs) (right; n=4). Bars indicate median values. *Statistically significant difference, paired t-test. (b) Western blots showing TSLP protein expression in lysates of oral mucosal epithelial sheets (ES; n=4) and cultured oral KCs (n=4) by use of either polyclonal anti-TSLP antibody (TSLP pAb) (ab47943) (left), with the same antibody absorbed with sfTSLP peptide (sf abs TSLP pAb; middle) or with rabbit immunoglobulin G (IgG) (isotype control, right; n=3). Numbers indicate the positions of the molecular weight markers. Black arrowhead indicates position of sfTSLP. (c) Expression of sfTSLP mRNA (left) and protein (right, n=5) in cultured oral keratinocytes transfected with scramble endoribonuclease-prepared siRNA (esiRNA) or with TSLP-specific esiRNA. Band density of detected bands in the scrambled control and TSLP esiRNA-treated samples (right, mean±s.d.; normalized to scrambled control). *Statistically significant difference, paired t-test. (d) Immunohistochemical staining (brown color) of sections of oral mucosa and skin by use of either TSLP pAb (ab47943) (far left) or the same antibody absorbed with sfTSLP peptide (middle left; representative stainings, n=10 for oral mucosa, n=5 for skin). Counterstaining with hematoxylin. In situ hybridization by use of sfTSLP-specific probe (middle right; representative stainings, n=5 for oral mucosa, n=3 for skin) or scramble mRNA probe (far right) on sections of oral mucosa and skin. Counterstaining with nuclear fast red. Pictures were taken at an original magnification of × 20. Bar=100 μm. (e) Relative mRNA expression of sfTSLP (n=8 for polyinosinic-polycytidylic acid (poly(I:C)), n=5 for interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α)/interleukin-1β (TNFα/IL-1β)) and lfTSLP (n=9 for poly(I:C), n=6 for IFN-γ and TNFα/IL-1β) in cultured oral KCs in response to the indicated stimuli. Cells were stimulated for 4 h and overnight (ON). Bars indicate median values. *Statistically significant difference, repeated-measures analysis of variance on ranks with pairwise multiple comparison. (f) Expression of sfTSLP at 9 kDa (ab47943) (left, n=3) and lfTSLP at 23 kDa (no. 4021) (right, n=4) protein in response to poly(I:C) for 4 h and ON in cultured oral keratinocytes. Colored numbers and colored arrows indicate the positions of the molecular weight standards. Black numbers and black arrows indicate the positions of sfTSLP and lfTSLP. (g) Immunohistochemical staining (brown color) of sections of oral mucosa exposed to smokeless tobacco “snus,” by use of either TSLP pAb (ab47943) or the same antibody absorbed with sfTSLP peptide (representative stainings, n=5). Counterstaining with hematoxylin. Pictures were taken at an original magnification of × 10. Bar=100 μm. sf abs TSLP pAb, sfTSLP absorbed anti-TSLP pAb. (h) Immunohistochemical staining (brown color) of sections of submandibular gland made by use of either TSLP pAb (ab47943) (left) or the same antibody absorbed with sfTSLP peptide (middle) (n=3). Counterstaining with hematoxylin. In situ hybridization sfTSLP-specific probe (right; blue color; n=3). Counterstaining with nuclear fast red. Pictures were taken at an original magnification of × 20. Bar=100 μm. (i) Detection of sfTSLP in saliva by western blotting. A specific band at 9 kDa (black arrow) was detected by use of TSLP pAb (no. 4021) (left), which was not visible after use of the same antibody absorbed with sfTSLP peptide (right) (n=8). Colored numbers and colored arrows indicate the positions of the molecular weight standards.
