4.11.2. Screening of the Cell Lines by Western Blot

To avoid the use of trypsin, cells were lysed in situ with a 500 µL RIPA lysis buffer supplemented with protease-inhibitors (RIPA lysis buffer system, sc-24948, Santa Cruz Biotechnologies) at 4 ◦C for 30 min. Lysates were clarified by centrifugation at 12,000× *g* at 4 ◦C for 15 min, and then protein concentrations were measured using the DC Protein Assay (Bio-Rad). About 40 µg of proteins were separated using Bolt Bis-Tris Plus precast 8% polyacrylamide gels with an MOPS SDS running buffer (Thermo Fisher) then blotted onto PVDF membranes. After blocking with TBS containing 0.1% Tween 20 and 5% BSA for 2 h at room temperature, and membranes were incubated overnight at 4 ◦C with (antihuman CCK-2R) mouse monoclonal antibodies conjugated with phycoerythrin (clone E-3, sc-166690, Santa Cruz Biotechnology) diluted 1:400 in TBS containing 0.1% Tween 20 and 5% BSA. Fluorescent signals were detected with the ChemiDoc instrument (Bio-Rad) using a Cy3 filter. To check the protein load, membranes were afterwards incubated overnight at 4 ◦C with rabbit anti-human GAPDH antibodies (Santa Cruz Biotechnologies) diluted 1:500 in TBS containing 0.1% Tween 20 and 5% BSA. Signals were detected by 1-h incubation at room temperature with HRP-conjugated secondary antibodies (Abcam, AB6013), followed by incubation with an ECL detection reagent (Thermo Fisher) and ChemiDoc imaging (Bio-Rad).
