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. 2015 Mar 28;6(18):16084–16105. doi: 10.18632/oncotarget.3177

Figure 7. DDR1 level affects IGF-IR protein expression.

Figure 7

(a) IGF-IR protein expression after siDDR1 silencing. Breast cancer cells were transiently transfected with siRNA to DDR1 or scramble siRNAs. After 72 h, cells were lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. Blot is representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments. Statistical significance was determined using Student's t-test. NS, p > 0.05; *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001; (scramble vs. siDDR1 conditions). (b) IGF-IR protein expression after DDR1 overexpression. Breast cancer cells were transiently transfected with plasmids encoding either the human wild-type DDR1 (DDR1/wt), or the corresponding empty vector (EV). After 72 h, cells lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used to control for protein loading. The top panels show a representative experiment. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. Statistical significance was determined using Student's t-test. NS, p > 0.05; *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001; (EV vs. DDR1 transfected cells). (c) IGF-IR protein expression and downstream signaling after DDR1 overexpression in R+cells. R+ fibroblasts stably transfected with plasmids encoding either DDR1/wt or the DDR1/K618A mutant or the corresponding empty vector (EV), were lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used to control for protein loading. The top panel shows a representative experiment. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. Statistical significance was determined using Student's t-test. NS, p > 0.05; *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001; (EV vs. DDR1/wt or DDR1/K618A). (d) IGF-IR mRNA levels after DDR1 silencing in R+cells. IGF-IR mRNA levels were evaluated in the same cells shown in (C) Normalization was done using β-actin as housekeeping control gene. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined using one-way ANOVA. NS, p > 0.05; (EV vs. DDR1/wt vs. DDR1/K618A). (e) IGF-IR protein expression after DDR1 overexpression in R−cells. R− mouse fibroblasts stably expressing either the human DDR1/wt or the DDR1/K618A mutant or the corresponding empty vector (EV) were transiently transfected with plasmids encoding for either the IGF-IR/wt or the IGF-IR/K1003R mutant. 24 h after transfection, cells were lysed and analyzed by immunoblot.β-actin was used for control of protein loading. The panel shows a representative of three of independent experiments.