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. 2021 Jan 19;4:90. doi: 10.1038/s42003-020-01613-w

Fig. 4. LRIG1 and LRIG3 promote BMP signaling.

Fig. 4

a Different LRIG protein expression levels were induced in LRIG-inducible MEFs by treating the cells with different concentrations of the inducer doxycycline. Thereafter, the cells were stimulated with 2.5 ng/ml BMP4 for one hour followed by coimmunocytofluorescence analyses of nuclear pSmad1/5 and the FLAG-epitope present as a tag on the induced LRIG proteins. Correlation plots of phosphorylated Smad1/5 versus the FLAG-LRIG protein expression levels are shown. Fitted lines indicate the linear relationship with pSmad1/5 for each LRIG protein. Pearson’s correlation coefficients for the respective genes were LRIG1, 0.9162; LRIG2, 0.6939; LRIG3, 0.9251; and empty vector, 0.1659. Shown are three experimental repeats using three biological replicates. b Lrig-null MEFs were transfected with a full-length LRIG1-GFP fusion protein (LRIG1-GFP), an LRIG1-GFP fusion protein variant lacking the cytosolic domain of LRIG1 (LRIG1-Δcyto-GFP), or empty vector (GFP) as a transfection control. Thereafter, the cells were stimulated with 20 ng/ml of BMP4 for 20 min followed by coimmunocytofluorescence analyses of nuclear pSmad1/5 and the green fluorescence from GFP fusion proteins or control GFP. The correlation plots between pSmad1/5 and GFP fluorescence are shown. The fitted lines indicate the linear relationship to pSmad1/5 for the respective construct. Pearson’s correlation coefficients for the respective constructs were as follows: full length LRIG1, 0.8943; LRIG1-Δcyto, 0.9663; GFP control, 0.2564. Shown are two experimental repeats using three biological replicates. c Lrig-null MEFs were transfected with different amounts of expression vectors encoding FLAG-tagged full-length LRIG1 (LRIG1) or FLAG-tagged LRIG1 ectodomains (LRIG1-ecto). Thereafter, the cells were stimulated with 20 ng/ml of BMP4 for 20 min followed by coimmunocytofluorescence analyses of nuclear pSmad1/5 and the FLAG-epitope. Shown are the correlation plots between pSmad1/5 and FLAG-LRIG expression levels. Fitted lines indicate the linear relationship between pSmad1/5 and the respective FLAG-LRIG construct. Pearson’s correlation coefficients for the respective constructs were as follows: full-length LRIG1, 0.7393; and LRIG1-ecto, 0.5287. Shown are two experimental repeats using three biological replicates.