Fig 2: Binding and cellular incorporation of B2RAs in MDA-MB-231 cells(A) MDA-MB-231 cells were incubated with FITC-labelled peptide HOE140 or NG68 (10 µM) at 37°C for the indicated times, washed with PBS buffer, fixed with paraformaldehyde then analyzed by laser confocal microscopy. A pseudocolor fluorescence intensity scale is shown on the right; black being the lowest and white the highest value. Note the rapid cellular incorporation of FITC-NG68 peptide and its perinuclear/nucleolar localization increasing over time while FITC-HOE140 conserved an exclusive membrane distribution. No signal was detected when cells were incubated with FITC alone under the same experimental conditions. Individual midsection confocal images are representative of three replicates. (B) Immunocytochemical localization of B2R in MDA-MB-231 cells was performed in unpermeabilized (upper panels) and permeabilized (bottom panels) conditions using the B2R antiserum AS276–83. An Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody was used as a secondary antibody. Nuclei stained with Hoescht 33342 (middle panels, arbitrary colors). Merge in right panels. Negative control: no primary antibody. Pseudocolor fluorescence intensity scale as in A. Representative confocal middle z-sections are shown.

Fig 3: IHC detection of endogenous AltB2R and B2R in human breast and prostate biopsies. Representative images of IHC staining of breast (A) and prostate (B) cancer cases for B2R, AltB2R, and nonimmune IgG. Corresponding H&E images are shown. Advanced stages of breast and prostate cancer tissues showed relatively high levels of B2R and AltB2R immunoreactivities than normal tissues. Scale bar: 100 µm.

Fig 4: AltB2R and B2R are coexpressed from the same coding sequence and colocalize in the cytoplasm and membranes of HeLa cells.A, strategy used to detect AltB2R and B2R. B2R with an in-frame C-terminal Flag tag, without and with an out-of-frame C-terminal HA tag on AltB2R within the coding sequence of B2R, and AltB2R with an in-frame C-terminal tag without the reference B2R. Positions of start and stop codons are indicated in green and red, respectively. Construction drawings are not to scale. B, detection of B2RFlag and AltB2RHA by western blotting using anti-Flag and anti-HA antibodies. Tubulin serves as a loading control and NPTII serves as a transfection control. Representative western blot of two independent experiments performed. C, subcellular localization of both B2RFlag and AltB2RHA by immunofluorescence using primary antibodies as in B. B2RFlag (green) and AltB2RHA (red) staining is shown in punctate granular vesicle-like structures within cytoplasm, perinuclear area, and underneath the plasma membrane. Scale bar: 10 µm. D, expression of B2R with an N-terminal Flag tag (FlagB2R) in live, nonpermeabilized cells confirmed localization at the plasma membrane. As expected, no immunofluorescent labeling of intracellular AltB2RHA was observed in nonpermeabilized conditions. Scale bar: 10 µm. C and D, representative single confocal images (midsection of cells) from three independent experiments. E, subcellular fractionation shows an enrichment of AltB2RHA in the cytoplasmic fraction compared with the whole cell lysat (WCL). Immunoblots of CD71 (plasma membrane; Mem), Lamin A/C (nucleus; Nuc), and GAPDH (cytoplasm; Cyto) were used to assess the purity of each fraction. Representative western blot of two independent experiments performed.

Fig 5: HEK293A cells transiently transfected with B2R(Ø)mutant (lacking AltB2R) show no difference in receptor internalization kinetics but show decreased G protein coupling efficiencies following agonist stimulation.A–C, G protein coupling efficiency after stimulation with NG291 in HEK293A cells transfected with B2R and B2R(Ø), by following BRET proximity of Gaq-RLuc2 and GFP10-G?1 (A), Gai2-RLuc2 and GFP10-G?1 (B), or Gai3-RLuc2 and GFP10-G?1 (C). Data represent mean ± SD, n = 3, 4, 4, unpaired t-test for EC50 (not significant) and Emax, †p < 0.05, ††p < 0.01. D, ß-arrestin2 recruitment at the plasma membrane after stimulation with NG291 in HEK293A cells transfected with B2R and B2R(Ø) by following BRET proximity of ß-arr2-RLuc2 and rGFP-CAAX. Data represent mean ± SD, n = 3, unpaired t-test for EC50 and Emax, not significant. E and F, internalization kinetics of B2R by cell surface ELISA after stimulation with 1 µM RMP-7 (E) or NG291 (F) in HEK293A cells transfected with FlagB2R or FlagB2R(Ø), respectively. Note. B2R-HEK293A and B2R(Ø)-HEK293A cells gave equivalent signals at time zero (unstimulated) (not shown). Data represent mean ± SD, n = 3, 3, multiple comparison versus corresponding time point using two-way ANOVA with Sidak's correction, unpaired t-test for Imax (maximal internalization) and half-life, not significant.