Localization of B4GALNT2 and its role in mouse embryo attachment Pei-Tzu Li, M.S., Chi-Jr Liao, M.S., Lung-Chi Yu, Ph.D., Wen-Guey Wu, Ph.D., Sin Tak Chu, Ph.D.  Fertility and Sterility  Volume 97, Issue 5, Pages 1206-1212.e3 (May 2012) DOI: 10.1016/j.fertnstert.2012.02.019 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Cellular localization of B4GALNT2. (A) After transfection of GFP (upper panel) or B4GALNT2-GFP (lower panel) cDNA into 293T cells, confocal microscopy was used to view the cells to determine the localization of the transmembrane protein. Scale bar = 100 μm. (B) Immunofluorescence staining performed with a control IgG instead of anti-B4GALNT2. (C) and (D) Immunofluorescence staining performed with and without permeabilization, respectively. A blastocyst from the uterus of an E3.5 pregnant mouse was incubated with anti-B4GALNT2 and then immunolabeled with FITC-conjugated rabbit anti-IgG. The blastocyst then was viewed using confocal laser microscopy. A total of 100 blastocysts were analyzed in each experimental group. The confocal staining pattern shown here is representative of all blastocysts analyzed. The upper panel indicates the location of B4GALNT2. The lower panel shows nuclear staining with DAPI. Fertility and Sterility 2012 97, 1206-1212.e3DOI: (10.1016/j.fertnstert.2012.02.019) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Localization of B4GALNT2 in uterine tissue from pregnant mice. Uterine tissue was collected at different time points during pregnancy and on P1. (A) Gene expression in uterine tissue and blastocysts was measured by real-time quantitative PCR at different time points. The results are represented as the mean ± SD for each group. Note that the SD for the blastocysts is too small to view. (B) Western blot analysis was used to determine protein localization within the uterine tissue: m, plasma membrane fraction; c, cytosolic fraction. GAPDH served as the cytosolic internal control; Na/K-ATPase served as the plasma membrane internal control. The experiments were repeated 3 times. Fertility and Sterility 2012 97, 1206-1212.e3DOI: (10.1016/j.fertnstert.2012.02.019) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 In vitro and in vivo embryo attachment assays. (A) In vitro attachment assay. Twenty E3.5 embryos from each treatment group were harvested and incubated with confluent endometrial cells (i.e., Ishikawa cells) for 48 hours (equal to E5.5). Natural (N) and Tyrode's solution/assisted (T) hatching was carried out in the presence of 10 μg anti-B4GALNT2 (light gray bars) or 25 μg anti-B4GALNT2 (dark gray bars) or without antibody (black bars). (B) In vivo implantation assay. Pregnant mice were administered 10 or 25 μg anti-B4GALNT2 at E3.5, and the number of implanted embryos at E10.5 was counted. Controls included no antibody administration (Con) and no PBS administration (0). The experiments were repeated at least 4 times and presented as the mean ± SEM for each group. ∗∗P<.01; ∗∗∗P<.001. Fertility and Sterility 2012 97, 1206-1212.e3DOI: (10.1016/j.fertnstert.2012.02.019) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Inhibition of in vitro embryo attachment by lectins. Increasing concentrations of the lectins DBA, ConA, and UEA-1 (0–20 μg/mL) were added to the medium used for the in vitro embryo attachment assay (black bars). The synergistic effects of the lectins in combination with the anti-B4GALNT2 antibody (ab; 10 μg) are shown in gray bars. The results are presented as the mean ± SEM for each group. ∗∗P<.01; ∗∗∗P<.001 (n ≥ 3). All of the experiments were compared with the control. (B) Immunofluorescence staining performed with FITC-conjugated DBA. Blastocysts from the uteri of E3.5 and E4.5 pregnant mice were incubated with FITC-DBA for 2 hours and then observed under confocal laser microscope. The staining pattern shown here is representative (n ≥ 10). Fertility and Sterility 2012 97, 1206-1212.e3DOI: (10.1016/j.fertnstert.2012.02.019) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Proposed structure of B4GALNT2. (A) The predicted outside, inside, and transmembrane domains of B4GALNT2 based on results using the Hidden Markov Model (18). The red region indicates the transmembrane domain of the molecule. (B) Hydrophobicity analysis of the B4GALNT2 protein sequence, conducted according to the methodology of Kyte and Doolittle (29). A positive score indicates the hydrophobic domain of the molecule. Fertility and Sterility 2012 97, 1206-1212.e3DOI: (10.1016/j.fertnstert.2012.02.019) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 In vitro embryo attachment assay (AsPC-1). The pancreas adenocarcinoma cell line, AsPC-1, was used in the in vitro embryo attachment assay as a control. The blastocysts were transferred to confluent AsPC-1 cells or Ishikawa cells. After 48 hours, the adhesion rate was calculated as a percentage of attached blastocysts. (∗∗∗P<.001 vs. Ishikawa cells). The experiment was repeated 3 times. Fertility and Sterility 2012 97, 1206-1212.e3DOI: (10.1016/j.fertnstert.2012.02.019) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Proposed mechanism whereby B4GALNT2 is involved in embryo implantation. After B4GALNT2 expression in the endometrial epithelium and blastocyst, post-translational modification produces Sda-conjugated molecules and provides ligands for association with plasma-membrane–anchored B4GALNT2 on the blastocyst or another Sda antigen acceptor on the endometrial epithelia. The question marks indicate the possibilities of Sda antigen and its receptor. Fertility and Sterility 2012 97, 1206-1212.e3DOI: (10.1016/j.fertnstert.2012.02.019) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions