Fig 1: Altered expression of SP-D mRNA and protein in the early and late stages. (A) qRTPCR of SP-D transcript in 2M Control (n= 4), Early TRAMP (n=5), 6M Control (n=5) and Late TRAMP (n=8) normalized with GAPDH. (B) Representative images of SP-D immunolocalization. (C) Semi-quantitative immunohistochemical (IHC) analysis of SP-D protein in TRAMP (early and late) and age-matched control mice. The intensity of SP-D staining was quantitated by an open source plugin, IHC profiler compatible with open resource digital image analysis software, Image J followed by calculation of H score. For each tissue section, a minimum of three to four areas were scanned and average H score was calculated. The bar graphs represent the average H score of 2M Control (n= 3), Early TRAMP (n=3), 6M Control (n=4), Late TRAMP (n= 5) tissue samples. Inner boxes represent negative staining. Statistical significance was tested using one-way ANOVA followed Tukey’s multiple comparisons test (**p < 0.01). The data presented are mean ± SEM. ns, Non-significant.
Fig 2: Impaired expression of the intact form of SP-D throughout the tumour progression. Total protein lysates were analysed by Western blot for SP-D and GAPDH. (A) Immunoblot analyses of intact monomeric form of SP-D (~45kDa) and cleaved SP-D fragments (~35kDa-~33kDa) in the Early TRAMP compared with the corresponding 2M controls. C2: 2M Control mice; ET: Early TRAMP; 3 C2: 2M Control mice no. 3; 4 C2: 2M Control mice no. 4; 2089 ET: Early TRAMP mice no. 2089; 2090 ET: Early TRAMP mice no. 2090; 2091 ET: Early TRAMP mice no. 2091. (B) Immunoblot analyses of intact monomeric form of SP-D (~43kDa), differentially glycosylated form of SP-D (~50kDa) and cleaved SP-D fragments (~35kDa-~33kDa) in the Late TRAMP, compared with the corresponding 6M controls. C6: 6M Control mice; LT: Late TRAMP; 6 C6: 6M Control mice no. 6; 7 C6: 6M Control mice no. 7; 8 C6: 6M Control mice no. 8; 9 C6: 6M Control mice no. 9; 2059 LT: Late TRAMP mice no. 2059; 2038 LT: Late TRAMP mice no. 2038; 2063 LT: Late TRAMP mice no. 2063; 2061 LT: Late TRAMP mice no. 2061. GAPDH was used as a loading control. (C–G) Densitometric analysis of proteins normalized with GAPDH. n=3-6 for each group. Data are represented as mean ± SEM. Statistical significance was determined using two-tailed Student’s t-test (C); one-way ANOVA followed Tukey’s multiple comparisons test (D–G), NS, Non-significant. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
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