Reversed phase ziptip

Twenty million RL95‐2 cells were used per immunoprecipitation. Two independent experiments were performed with two technical replicates per immunoprecipitation. For each immunoprecipitation, 30 µL of protein G sepharose 4 fast flow (GE Healthcare 17061801) was bound to 5 µL of anti‐HN1 (GeneTex GTX106585) for 2.5 hours at 4°C with end‐over‐end rotation. Cells were lysed in 50 mmol/L Tris (pH 7.4), 137 mmol/L NaCL, 10% glycerol, and 0.1% Triton X supplemented with 1 X Halt protease and phosphatase inhibitor with 5 mmol/L EDTA (ThermoFisher Scientific 1861280) for 30 min on ice. Lysate was spun at 16 000 × g for 20 minutes at 4°C and the supernatant was incubated with protein G sepharose 4 fast flow and anti‐HN1 for 3 hours at 4°C with end‐over‐end rotation. Sample was resolved on a 4%‐15% mini‐PROTEAN TGX gel (Bio‐Rad 4561084) and 10 gel bands per lane were cut. Gel bands were digested using trypsin/LysC Mix (Promega V5073) overnight and samples were cleaned up using Reversed‐Phase ZipTip (Millipore ZTC185096) according to the manufacturer's protocol prior to LC‐MS/MS analysis. Immunoprecipitation with anti‐JPT1 and protein G sepharose 4 fast flow was performed in duplicate. Immunoprecipitation with protein G sepharose 4 fast flow alone was used as a control.

Molecular masses of intact peptides and the products of Mmp1 degradation were determined (CEQUIBIEM, University of Buenos Aires). Samples were desalted through reversed-phase ZipTip (Millipore, MA) following manufacturer's instructions and analyzed on an Ultraflex II MALDI TOF TOF (Bruker Daltonics) in Reflectron Positive mode and Lift mode using standard instrument settings, and HCCA matrix.