Journal: Nature cell biology
Article Title: Lrig1 controls intestinal stem cell homeostasis by negative regulation of ErbB signalling
Figure Lengend Snippet: Lrig1 controls endogenous signalling via the ErbB pathway a) Loss of Lrig1 causes increased protein levels and activation of the ErbB pathway. pEgfr, Egfr, pErbB2, ErbB2, pErbB3, ErbB3, cMet were detected by Western blotting in samples enriched for intestinal epithelium. β-actin is used a loading control. b) Relative expression analysis of the receptors by qPCR at P10 shows minor differences. Expression levels are shown relative to control samples (KO/Ctrl). Asterisks indicate significant changes ( ErbB2 : p=0.004; ErbB3 : p=0.04; KO n=4, Ctrl n=3). c-f) Increased activation of MAPK signalling and cMyc signalling upon loss of Lrig1. Immunohistochemical analysis for p-MEK1-2 (c-d) and cMyc (e-f). g-i) Altered Egfr activation dynamics upon loss of Lrig1 KO. i) Average normalised membrane intensity of pEgfr in intestinal samples from KO (n=6) and control animals (n=10) for 6-18 individual crypts per sample. Error bars represent the s.e.m. (positions 1-4: p
Article Snippet: Antibodies The following antibodies were used rabbit anti-β-catenin (Santa Cruz, sc-7199, 1:250), rabbit anti-Phospho-histone H3 (ser10) (Cell Signaling #9701, 1:400), rabbit anti-phospho-MEK1/2 (Ser221) (Cell signalling, #2338, 1:500), mouse anti-BrdU (Cell signaling, Bu20a, 1:250), goat anti-Lrig1(R and D Systems, AF3688, 1:100), rabbit anti-Myc (Millipore, 06-340, 1:100), mouse anti-Ki67 (Monosan, Clone MM1, 1:1000), PE-conjugated anti-Lrig1 (R and D systems, FAB3688P, 10μL/106 cells), Pacific blue and Alexa647 conjugated Rat anti-CD24 (Biolegend, M1/69, 5μL/106 cells), rat anti-Msi1 (MBL, D270-6, 1:10000), rabbit anti-phospho Egfr (Y1068) (Abcam, EP7774Y, 1:250), rabbit anti-Egfr (Cell Signaling, #4267 clone D38B1, 1:250), rabbit anti-phospho ErbB2 (Y1248) (Abcam, ab47755), mouse anti-ErbB2 (Millipore, 05-1130, Clone N3D10, 1:500), rabbit anti-phospho ErbB3 (Y1289) (Cell Signaling, #4791, Clone 21D3, 1:250), mouse anti-ErbB3 (Millipore, 05-390, Clone 2F12, 1:500), rabbit anti-cMet (Santa Cruz, sc-162, 1:500), mouse anti-β-actin, (Sigma A5316, clone AC-74, 1:5000) and Atto-488 conjugated UEA-1 (Sigma, 10μL/106 cells for flow cytometry, 1:1000 for near-native sections) according to manufacturer’s instructions.
Techniques: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry