Digital imaging

Lipoplexes were assessed against DNase I degradation as follows:
1. adding 2 units of DNase I to each intact lipoplex formulation containing 1 μg pDNA and 20 μg liposomes/100 μl buffer, to give ratio of 1:20 DNA:liposomes, at 37 °C for 30 min. 10 μL from each sample was run in gel electrophoresis to measure the protection that liposomes provide for pDNA. This can also be used to measure if pDNA has been encapsulated inside the liposomes, or is just bound to the liposome membrane.
2. Adding DNase I to the dissociated lipoplexes (20 μl of chloraphorm/methanol 2:1 were used in order to dissociate the lipid/DNA complexes before adding the DNase I). After 30 min, 10 μL from each sample was run in gel electrophoresis
3. 1 μg of pDNA was incubated with 2 units of DNase I in 100 μl buffer (this was used as a reference to compare with intact lipoplex formulations).
All samples run in 1% agarose gel for 60 min at 90 vm. Results were analysed by UV trans-illumiator with digital imaging (BioRad Laboratories, Inc).

Skimmed milk (5 µL) or MEC lysates (20 µg of protein) were prepared in Laemmli sample buffer containing 100 mM dithiothreitol (DTT), electrophoresed and immunoblotted as previously described^{29 (link)}. The following antibodies were used: anti-PRLR (1:1000, Abcam), anti-VAMP7 (1:1000, ThermoFisher Scientific), anti-SOX4 (1:1000, ThermoFisher Scientific), and anti-HA (1:1000; Roche Applied Scientific). Antibodies were detected with horseradish peroxidase-conjugated anti- rabbit or anti-mouse IgG (GE Healthcare). Membranes were stripped before re-probing with anti-HA as a normalization control. Protein was detected with SuperSignal Femto Chemiluminescent Detection System (Pierce) and imaged using digital imaging (BioRad). Band signal intensity was quantified using ImageStudio software (LICOR).

Cells were washed, lysed with lysis buffer [250 mM NaCl, 10% glycerol, 2mM EDTA, 0.5% IGEPAL (Sigma-Aldrich), 50 mM HEPES (Sigma-Aldrich)], and then centrifuged at 13,000 RPM for five minutes. Afterwards, 2x Laemmli sample buffer (Bio-Rad Laboratories, Hercules, California) was added to the lysate for a 1:1 dilution and the sample was denatured at 95 °C for five minutes. Then, the sample was loaded onto a 4–20% gradient Mini-PROTEAN TGX precast gel (Bio-Rad Laboratories) and electrophoresed at 100 V for 70 minutes, before being transferred to a PVDF membrane (Bio-Rad Laboratories) via wet transfer. Membranes were blocked with 5% dry milk in TBST [10 mM Tris-HCl, 100 nM NaCl, 0.1% Tween 20] for 1 hour and then incubated with an anti-GFP primary antibody (Abcam) at a 1:5000 dilution or a GAPDH primary antibody (Santa Cruz, Dallas, TX) at a 1:3000 dilution overnight at 4 °C. Afterwards, the membrane was rinsed three times with TBST and incubated with the appropriate species-specific enzyme-conjugated secondary antibody (Life Technologies), for 1 hour at room temperature. Membranes were later developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The resulting signal was detected using digital imaging (Bio-Rad Laboratories).