Nature research paper: Mechanism-based traps enable protease and hydrolase substrate discovery
for 1 h. The membrane fraction was dissolved in 2% SDS buffer . The solution was diluted by 10% NP40 to generate a final concentration of 0.1% SDS and 1% NP40. One-hundred microlitres MagStrep type3 XT beads were added to the solution and incubated at room temperature for 1 h. The beads were washed with RIPA and PBST three times each. Proteins attached to the beads were eluted in 1× LDS loading buffer by heating at 65 °C for 15 min.
To analyse proteins in the extracellular medium, FBS-containing medium for HEK293T cells was replaced with hybridoma serum free medium 24 h before collection. Expi293 medium, which is serum-free and protein-free, can be directly collected for further analysis. The medium was collected and filtered through a 0.22 μm polyethersulfone membrane. To obtain total proteins in the medium, 1/10 volume of 100% ice-cold TCA solution was added at 4 °C for protein precipitation.
Deglycosylation was performed by adding 1/10 volume of 10% NP40 and PNGase or DeGlycosylation mix II to proteins dissolved in the 1× LDS loading buffer. The reaction was incubated at 37 °C for 1 h before analysis.Twenty-four hours after transfection, Expi293 cells were split into two halves treated separately with DMSO or BFA .
To detect the proteolytic fragments from endogenous substrates generated by endogenous RHBDL4, 10 million wild-type or RHBDL4 knockout HCT116 cells were cultured in hybridoma serum free medium for 40 h. The medium was collected, filtered and concentrated by a 30 kDa cut-off concentrator. Proteins were precipitated by TCA and dissolved in 1× LDS loading buffer for immunoblotting analysis.
To ensure cryo-protection, crystal-containing drops were mixed with 25% glycerol in reservoir solution prior to picking and flash frozen in liquid nitrogen. Diffraction data was collected at the Diamond Light Source on beamline I04. Datasets were auto-processed with XIA2 DIALS , scaled using Aimless and Refmac5 in the CCP4 suite of programs. Structure refinement and manual model building were performed with Refmac5 and COOT . Colour figures were prepared with PyMol .
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