Clone lm609

Cells were grown on cover glasses for 1 day, rinsed with TBS at room temperature and fixed for 10 min with 4% paraformaldehyde. After rinsing with TBS, in the case of permeabilized conditions, cells were incubated with 0.1% Triton X-100 in TBS for 30 min followed by 30 min in blocking buffer (BSA 2% in TBS-T 0.1% Triton X-100) at room temperature. For non-permeabilized conditions, cells were directly incubated in blocking buffer (BSA 2% in TBS) for 1 h at room temperature. Next, cells were incubated in the suitable primary antibodies for 1 h at room temperature and rinsed repeatedly with TBS before incubating in the appropriate fluorescein-labelled secondary antibody for 1 h at room temperature. Cells were then washed extensively with TBS and mounted on a glass slide in Prolong Diamond Antifade mountant with DAPI (Pierce, Thermo Fisher Scientific). The following primary antibodies were used: anti-EEA1 (1:1000) (Cell Signalling), anti-LAMP-1 (1:1000) (Santa Cruz), anti-avb3 (1:50) clone LM609 (Merck), and anti-tubulin (1:1000) (DM1a-FTIC, Sigma). The following secondary antibodies were used: goat anti-mouse IgG (H + L) Alexa Fluor Plus 488 and goat anti-rabbit IgG (H + L) Alexa Fluor 633 (Thermo Fisher Scientific) (1:1000).

Cells were trypsinized into a single cell suspension before staining. The primary antibodies were mouse anti-human αvβ3 (1 μg/sample) (Clone LM609 Cat no. MAB1976, EMD Millipore, Billerica, MA), mouse anti-human αvβ5 (1 μg) (Clone P1F6, Cat no. MAB1961 EMD Millipore), rat anti-mouse αv (1 μg) (Clone RMV7, Cat no. 50-104-13, eBioscience), rabbit anti-human NRP-1 b1b2^{20 (link)} (1 μg), and Alexa 647 or BV421-conjugated mouse anti-human/mouse αvβ5 (1:50) (clone ALULA Cat no. 565836 or 743669, BD Biosciences, San Jose, CA). The primary antibodies were detected with corresponding secondary antibodies conjugated with Alexa 488, 594, or 647 (1:250) (Invitrogen). The cells were analyzed with an LSR Fortessa System (BD Biosciences), and the data were analyzed with a FlowJo software. Cells were gated on single cells and then on live cells by excluding DAPI or PI positive cells. In tumor cell/CAF co-culture experiments, mCherry high cells were gated in order to define the mCherry-labeled CAFs. For iRGD-AgNP uptake quantification, specific uptake was defined by gating cells positive for either Alexa 647 or 488, which were attached to the AgNPs.

A recently developed human recombinant anti-HPA-1a IgG1 mAb (clone 26.4) [16 (link)] was used to explore the effect on invasive trophoblast cells. Murine anti-human αVβ3 mAb, clone LM609 (Millipore, Billerica, MA) was used as positive control for cell functional studies. Sodium azide from LM609 sample was removed by buffer exchange with PBS using PD SpinTrap G-25 (GE Healthcare, Little Chalfont, UK). Integrin β3 was detected using murine mAb, clone SZ21, HPA-1-reactive [19 (link)] (Dako, Glostrup, Denmark) and rabbit mAb, clone EPR2417Y (Abcam, Cambridge, UK). Alexa Fluor 488-conjugated goat anti-mouse and goat anti-human antibodies (Invitrogen, Carlsbad, CA) were used as secondary antibodies in flow cytometry experiments. Human myeloma plasma IgG1 (Sigma) and murine IgG1 (Beckman Coulter, Brea, CA) were used as isotype controls. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Thermo Scientific, Waltham, MA) was used as a detection antibody in the western blot experiment.