Pierce ecl plus reagent

For protein electrophoresis, equivalent total protein was loaded into each lane of a 4–12% SDS-PAGE gel and run under reducing conditions. Resolved proteins were transferred to nitrocellulose membrane (GE Healthcare, Little Chalfont, United Kingdom), which was then blocked in 5% milk/TBST followed by overnight incubation in 5% milk/TBST supplemented with anti-PAD4 (ThermoFisher, Waltham, MA) or anti-PAD2 (Abgent, San Diego, California) antibody at 1:3000 or 1:1000 dilution, respectively. Membranes were washed in TBST and incubated for 1 hour in 5% milk/TBST buffer containing 1:4000 dilution of goat anti-rabbit HRP-conjugated secondary antibody (Abcam, Cambridge, UK). After final washes in TBST and TBS, blots were processed with Pierce ECL Plus reagent (ThermoFisher, Waltham, MA) and imaged using the Odyssey Fc imaging system (LI-COR Biosciences, Lincoln, NE). All blots are shown as single images with brightness and contrast adjusted to maximize visibility of the background and any non-specific bands.

Total tissue lysates were prepared in SDS-PAGE lysis buffer (150 mM NaCl; 100 mM Tris-HCl, pH 8.0; 12.5 mM EDTA; 2% SDS; 1% Triton-X100; 0.5% Nonidet-P40; 10% glycerol) containing Complete protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail II (Boston BioProducts, Inc., Ashland, MA) that were added to the lysis buffer in the amounts recommended by the suppliers. Samples were further disrupted with a Model 505 sonic dismembrator (setting 3 for 15 sec) (Thermo Fisher). Protein concentrations were then determined with a Pierce BCA Protein Assay Kit (Thermo Fisher). Depending on the proteins under study, 10–50 μg of lysate were used for SDS-PAGE and western blotting to PVDF membranes (Thermo Fisher, Inc.) as previously described [17 (link)]. All membranes were blocked for at least two hr. at 4°C in PBS containing 0.1% Tween (PBS-T) and 5% non-fat dry milk. Antibodies, vendors and conditions used for immuno-blotting are shown in Table A in S1 File. Most antibodies were incubated with membranes overnight. Following exhaustive washing in PBS-T, blots were then developed using a Pierce ECL Plus reagent (Thermo Fisher, Inc.) according to the directions provided by the supplier.

Twenty microgram of protein from whole‐cell lysate or 15 μL of eluted proteins from immunoprecipitation were separated by Tris/glycine SDS/PAGE (8% α‐actinin, 10% tubulin or actin, 12% SIKE), and transferred to a nitrocellulose membrane. The membrane was blocked in either 5% milk or 5% BSA (rabbit α‐tubulin‐HRP) in TBS/Tween. The immunoblot was probed for SIKE using mouse anti‐FLAG‐HRP or mouse anti‐HA or rabbit anti‐SIKE or cytoskeletal protein using either mouse‐α‐GFP (BioLegend) or rabbit α‐tubulin‐HRP (Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. The blot was then washed with TBS/Tween: 6 × 5 min (rabbit anti‐FLAG‐HRP) or 3 × 5 min (all other blots). For blots requiring a secondary antibody, goat anti‐mouse or goat anti‐rabbit IgG‐HRP (Southern Biotech, Birmingham, AL, USA), was incubated with blot for 1 h, and washed with TBS/Tween (4 × 10 min). See Table 2 for antibody dilutions. Membranes were developed with Pierce ECL Plus reagent (Thermo Fisher Scientific) as per manufacturer's protocol and documented on a Gel Doc XR+ gel documentation system (Bio‐Rad) or a c‐DiGit blot scanner (LI‐COR, Lincoln, NE, USA). For whole lysate immunoblots, the blots were stripped, blocked in 5% milk TBS/Tween, probed for actin using mouse anti‐actin‐HRP, washed with TBS/Tween (7 × 5 min), and developed as described above.

MCF10A and MCF10A CDH1-/- cells were grown for 72 h to 90% confluency in T25 flasks and lysed using cell culture lysis reagent (Promega) containing cOmplete mini protease inhibitor (Roche). BCA assays (Thermo) were performed to equalize total protein loaded. Proteins were separated on 10% SDS-PAGE gel for 2 h, followed by blot transfer at 100 V for 1 h. Immunoblotting was performed using rabbit anti-E-cadherin antibody (Santa Cruz, SC7870) at 1:200 dilution overnight, or rabbit anti-α-actin primary antibody (Sigma) at 1:1,500 dilution overnight followed by anti-rabbit HRP-linked secondary antibody (Santa Cruz) at 1:5,000 dilution for 1 h. Chemiluminescence was performed using Pierce ECLplus reagent (Thermo) and imaged using LAS-3000 (Fujifilm).