Igg h l

We used the following monoclonal and polyclonal capture and detection antibodies: PAPP-A (capture, Hytest, Turku, Finland; detection, R&D Systems, Minneapolis, MN, USA), fβ-hCG (capture, Acris Antibodies GmbH, Herford, Germany; detection, Hytest), AFP (both from Hytest), ANGPTL3 (both from R&D Systems), EGF (both from R&D Systems), IGFII (capture, Abcam, Cambridge, UK; detection, R&D Systems), SOD1 (capture, R&D Systems; detection, Hytest), and IgG (H + L) (Invitrogen, Breda, The Netherlands). Note that IgG was added for quality control purposes. Standards for PAPP-A and fβ-hCG were obtained from the AutoDELFIA kits. These standards were calibrated against the WHO International Reference Preparation (PAPP-A: 78/610 for SP1, fβ-hCG: 75/551). In addition to serum samples from the serum bank, the reference serum was used to correct for interarray variation.

Cell lysates were separated on a 10% tris-glycine polyacrylamide gel and transferred to a PDVF membrane at 100 V for 1 h. The membrane was blocked in 5% bovine serum albumin with PBS plus 1% Tween-20 (PBS-T) for 1 h while rotating, and washed 3 times with PBS-T. The membranes were incubated overnight at 4 °C with a c-Myc primary antibody at a 1 in 2000 dilution (Cell Signaling; D84C12), and washed 3 times with PBS-T before the addition of an anti-rabbit HRP polyclonal secondary antibody at a 1 in 4000 dilution (Invitrogen IgG (H + L); LS656120). Membranes were washed twice with PBS-T, once with PBS and imaged using HRP substrate (Luminata; WBLUF0100) in a BioRad Chemidoc. An uncropped version of the Western blot in Fig. 3b is supplied in the Source Data files.

Transwell membranes were processed for immunocytochemistry against Claudin-19 (1:100; R&D Systems), ZO-1 (1:100; Invitrogen), Ezrin (1:100; Cell Signaling Technology), MCT-1 (1:100; Sigma), CRALBP (1:100; Abcam), RPE65 (1:100; a gift from Dr. T. Michael Redmond, National Institutes of Health [NIH]), and nuclear stain DAPI (1:10,000; Invitrogen). Secondary antibodies used at 1:1,000 dilutions were Alexa Fluor 488-goat anti-mouse IgG2A, Alexa Fluor 546-goat anti-rabbit immunoglobulin G (IgG) (H⁺L), Alexa Fluor 488-goat anti-mouse IgG1, and Alexa Fluor 546-goat anti-mouse IgG (H⁺L) (Invitrogen). Transwell membranes were fixed with ice-cold 4% FA solution for 15 min and then blocked and permeabilized with a solution containing 0.1% saponin, 5% normal goat serum, and 1% bovine serum albumin in phosphate-buffered saline. Controls included incubations of secondary antibodies in the absence of primary antibodies and block solutions in the absence of primary/secondary antibodies. Fluorescent images were collected using a Leica TCS SP5 confocal microscope.