Radioimmunoprecipitation assay (ripa)

Cellular lysates were prepared in RIPA (Boston Bioproducts, Ashland, MA, USA) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Grand Island, NY 14072 USA). Protein was quantified using the BCA assay kit (Thermo Scientific, Grand Island, NY 14072), separated on SDS-PAGE gels and transferred to the Immun-Blot® PVDF membrane (Bio-Rad, Hercules, California 94547USA). Membranes were blocked in 5% nonfat milk and incubated with the following primary antibodies; BMI1 (Invitrogen at 1:1000 dilution), Fibronectin, N-Cad, E-Cad (BD Biosciences at 1:1000) EMA (Dako at 1:500 dilution), EPCAM, Snail, (Cell signaling technology at 1:1000 dilution), ATP7B (Protein Tech at 1:500) TWIST1, α tubulin and β actin (Sigma, TWIST1 at 1:1000, α tubulin and β actin at 1:10,000 dilutions), Secondary antibodies (from Sigma) were used at a concentration of 1:10,000.

Cells were harvested and lysed in lysis buffer (RIPA, Boston Bio Products) supplemented with Halt protease and phosphatases inhibitor cocktail (Thermo fisher Scientific). Protein concentrations were determined using Pierce™ BCA Protein Assay Kit (Thermo fisher Scientific). Equal amounts of protein (20–30 µg) were loaded on 10% or 4-12% Bis–Tris gel and transferred to PVDF or NC membrane followed by immunoblotting with 5% milk. Then, membranes were incubated with first antibody at 4°C overnight. After washed with Tris-buffered saline with Tween 20 (TBST) three times for 5 min, membranes were incubated with horseradish peroxidase (HRP)-conjugated second antibody for 1 h at room temperature, washed again with TBST three times for 5 min.

Total Cell Lysate was prepared in RIPA (Boston Bioproducts) or Cell Lytic M (Sigma) compatible with enzymatic assay and immunoprecipitation.
For the formalin fixed paraffin embedded (FFPE) patient sample, samples were prepared as previously described with slight modification [23 (link)]. First, samples were deparffinized with 1 ml of xylene; vortexed and stored for 10 min, followed by centrifugation at 14,000 g × 5 min and then supernatant was removed. Deparffinization process was repeated thrice following which samples were gradiently hydrated starting from 100% ethanol followed with 80% and 50% ethanol. After each step the samples were centrifuged at 14,000 g × 5 min and supernatant was removed. Samples were stored overnight at 4 °C in 1 ml of DEPC-treated water and then centrifuged at 14,000 g × 15 min and the pellet was resuspended in 2% SDS buffer with brief pulse of sonication (10 s × 3 times), centrifuged (14,000 g × 15 min) and supernatant collected for downstream assay.
Concentration of the extracted protein was determined by Bichonic acid assay (BCA) method using Pierce Kit (#23225). For most assays 10–30 μg of the lysate was used.
λ- Phosphatase treatment was performed on 50 μg of cellular protein incubated with 200units of the phosphatase at 25 °C for 1 h as per the manufacturer’s protocol. Reaction was terminated by addition of the 4 × lamelli buffer.