Supplementary Figure 5: EGF and BTC activate EGFR sequentially. | Nature Cell Biology

Supplementary Figure 5: EGF and BTC activate EGFR sequentially.

From: EGFR signalling controls cellular fate and pancreatic organogenesis by regulating apicobasal polarity

Supplementary Figure 5

(a) Representative section of E11.5 WT pancreata (6 μm) stained for mucin1 (Muc1;green), EGF (red), E-cadherin (Outlined;Not shown) and DAPI (blue). (b–b ′) Representative section of E15.5 WT pancreata (6 μm) stained for Neurogenin3 (Ngn3;green), mucin1 (Muc1;blue), EGF (red) and E-cadherin (E-cad; White (b); Outlined;Not shown (b ′)). Indicated region visualizes area of inset in b ′. (c) Representative section of E11.5 WT pancreata (6 μm) stained for mucin1 (Muc1;green), Betacellulin (BTC;red), E-cadherin (Outlined; Not shown) and DAPI (blue). (d–d ′) Representative section of E15.5 WT pancreata (6 μm) stained for Neurogenin3 (Ngn3;green), mucin1 (Muc1;blue), Betacellulin (BTC;red) and E-cadherin (E-cad; White (d); Outlined;Not shown (d ′)). Indicated region visualizes area of inset in d ′. (e) Representative section of E11.5 WT pancreata (6 μm) stained for mucin1 (Muc1;green), EGFR (red), E-cadherin (Outlined;Not shown) and DAPI (blue). (f–f ′) Representative section of E15.5 WT pancreata (6 μm) stained for Neurogenin3 (Ngn3;green), mucin1 (Muc1;blue), EGFR (red) and E-cadherin (E-cad; White (f); Outlined;Not shown (f ′)). Indicated region visualizes area of inset in f ′. (g) Representative section of E11.5 WT pancreata (6 μm) stained for mucin1 (Muc1;green), ErbB4 (red), E-cadherin (Outlined;Not shown) and DAPI (blue). (h–h ′) Representative section of E15.5 WT pancreata (6 μm) stained for Neurogenin3 (Ngn3;green), mucin1 (Muc1;blue), ErbB4 (red) and E-cadherin (E-cad; White (h); Outlined;Not shown (h ′)). Indicated region visualizes area of inset in h ′. (i) Representative western blot analysis of E11.5 or E12.5 explants treated with EGF or BTC (0, 10, 20 or 30 ng/ml) for 48 h, when added at E11.5 or E12.5 + 2, were analyzed for activation of EGFR (p-EGFR) during the primary or secondary transition. (Loading control: Vinculin). (j) Relative band intensity (p-EGFR/Vinculin) quantification from western blots in i. n = 3 protein samples, from two independent experiments, primary transition: EGF: 0/10 pTT = 0.1379, 0/20 pTT = 0.0029, 0/30 pTT = 0.2421; BTC: 0/10 pTT = 0.5939, 0/20 pTT = 0.1592, 0/30 pTT = 0.1247; secondary transition: EGF: 0/10 pTT = 0.7233, 0/20 pTT = 0.3370, 0/30 pTT = 0.2270; BTC: 0/10 pTT = 0.1050, 0/20 pTT = 0.0001, 0/30 pTT = 0.0017. Error bars represent ± SEM. Scale bar, 40 μm (a,c,e,g), 20 μm (b,b ′,d,d ′,f,f ′,h,h ′). Statistical analysis: two-tailed student’s t-test (pTT). Statistic source data are found in Supplementary Table 3.Unprocessed blots are shown in Supplementary Fig. 11.

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