Fig. 5: Effect of GPRC5B on EP2 expression and membrane availability.

A, B Ptger2 expression in RPM (A, n = 12)) and M0 BMDM (B, n = 15) was determined by qRT-PCR (normalization to Gapdh, control set to 1). C EP2 expression was determined by Western blotting in RPM and M0 BMDM lysates from control mice (Contr) and M-G5b-KOs (KO). GAPDH as loading control. D, E HEK cells co-transfected with HA-EP2 alone or HA-EP2 + GPRC5B-FLAG/Myc (G5B-Myc) were first subjected to plasma membrane staining with WGA, then fixed, permeabilized and stained with anti-Myc and anti-HA antibodies: exemplary photomicrographs (D) and statistical evaluation of the HA-EP2 signal within WGA (E) (n = 26 and 36 cells). F, G ELISA-based detection of EP2(ec) in the plasma membrane of RPM (F) and M0 BMDM (G) (n = 14(F), 16(G) samples from 3-4 mice per group). H, I Antibody-mediated detection of EP2(ec) in non-permeabilized RPM from control mice and M-G5b-KOs (CD11b staining indicates plasma membrane, global EP2-KOs shown as specificity control): exemplary photomicrographs (H) and statistical evaluation of EP2(ec) signal in CD11b area (I) (n = 44/37/7 cells from 3 mice per group). J, K RPM from control and KO mice were fixed, permeabilized and stained for EP2(ec) and Golgi marker GM130, and then stained with anti-CD11b antibodies (for plasma membrane): exemplary photomicrographs (J) and statistical evaluation (K) of the colocalization of EP2(ec) with CD11b, GM130 or calreticulin (examples for the latter in Supplementary Fig. 9C) (n = 14 cells from 2 mice each). Data are means ± SEM; differences between groups were analyzed using two-sided Mann-Whitney test (A, B, E), unpaired two-sided t-test (F, G), Kruskal-Wallis test with Dunn’s correction for multiple testing (I), unpaired two-sided t test with Holm-Sidak correction for multiple testing (K). n, number of analyzed mice (A, B, F, G) or cells (E, I, K); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are provided as a Source Data file.