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. 2019 Nov 15;2(6):e201900422. doi: 10.26508/lsa.201900422

Figure 2. LecB depletes IGF-1R from the plasma membrane without inducing its activation.

Figure 2.

(A) Western blot of eluted samples from pull-down assay. NHKs were stimulated in the presence of 5 μg/ml biotinylated LecB (106 nM). The lysates were incubated with streptavidin beads and further eluted. Western blot was performed and IGF-1R and LecB were detected in the precipitated fractions. (B, C) Surface staining of keratinocytes treated with LecB (5 μg/ml) for the indicated time points. (B) Images show maximum intensity projections. After stimulation, the cells were stained for IGF-1R (red) and DAPI (blue). Scale bar: 10 μm. (C) Quantification of IGF-1R surface intensity from n = 4 independent experiments. Bars display the mean value ± SEM. ** denotes P < 0.01; **** denotes P < 0.0001; one-way ANOVA was used for statistical analysis. (D) Representative blots from lysates after LecB (5 μg/ml) and IGF-1 (100 ng/ml) stimulation for the indicated times. Antibodies against different phosphorylation sites of IGF-1R (Tyr1131 and Tyr1135/1136) and against total IGF-1R were used. Tubulin was used as loading control. (E, F, G) Blot quantification. Phosphorylated IGF-1R (E, F) and total IGF-1R levels (G) are represented as fold change compared with the loading control from n = 3 independent experiments. * denotes P < 0.05; *** denotes P < 0.001; two-way ANOVA was used for statistical analysis.