Figure 1: GPRC5A post-transcriptionally downregulates EGFR expression.

(a) Flowchart of quantitative global proteomics using mTRAQ labelling. (b) Ratio distribution of Gprc5a^−/− mouse LBE cells relative to wild-type LBE cells. The ratios shift the overall distribution (—) relative to representative normal distribution (---). (c) Western blot of EGFR, GPRC5A and β-Actin in wild-type and Gprc5a^−/− LBE cells. (d) Semi-quantitative RT–PCR (left) and quantitative real-time PCR (right) of EGFR performed in triplicate using cDNAs obtained from wild-type and Gprc5a^−/− LBE cells. (e) Images of immunofluorescence (IF) for EGFR in the lung frozen section of wild-type and Gprc5a^−/− mice (top: Scale bar, 200 μm; bottom: Scale bar, 20 μm). (f) Real-time PCR of EGFR performed in biological triplicate using cDNAs obtained from wild-type and Gprc5a^−/− lung tissue. (g) Western blot of EGFR, GPRC5A and β-Actin in wild-type and Gprc5a^−/− LBE cells stably expressed with vector or GPRC5A (C1 and C2, respectively, represent Clone 1 and Clone 2). (h) Real-time PCR of EGFR performed in biological triplicate using cDNAs obtained from wild-type and Gprc5a^−/− LBE cells stably expressed with vector or GPRC5A (C1 and C2, respectively, represent Clone 1 and Clone 2). (i) The relative ratio of EGFR protein and mRNA (right) calculated by western blot (left) and real-time PCR (middle) of EGFR in wild-type and Gprc5a^−/− LBE cells stably expressed with vector or EGFR from three independent experiments (two-tailed Student’s t-test, ***P<0.001). All western blots were shown are from a single experiment that is representative of at least three biological replicates. All the data are mean with s.e.m. from biological triplicate.