Imagequant las 4000 gel imaging system

Samples were drawn through a cellulose acetate membrane (Sterilitech, Kent, WA, USA) and cellulose nitrate membrane (Bio-Rad, Hercules, CA, USA) using a Bio-Dot vacuum manifold (Bio-Rad). The membranes were pre-wetted with PBS, then 100 µL of sample was drawn through. The protein spots were washed 3 times by drawing 200 µL PBS through the membranes. Membranes were stained with 0.1% (w/v) amido black in 10% (v/v) acetic acid, then gently washed with 5% acetic acid and deionized water until the background stain was removed. Aggregated protein adheres weakly to cellulose acetate and can be knocked off by vigorous washing. Spots were imaged using an ImageQuant LAS 4000 gel imaging system (Cytiva) and quantitated with ImageLab software (Bio-Rad), treating each spot as a gel band in order to control the software’s background subtraction feature. The ratios between the background-corrected intensities are reported in Figure 5 and Figure 6 without further normalization.

LCs (5 µM) were incubated at 37 °C for 4 h in the presence of small molecules (50 µM) or appropriate vehicle control, with or without 200 nM proteinase K (Thermo Fisher). Reactions were initiated by adding 2 µL proteinase K or buffer to 18 µL LC solution and incubated in a thermocycler with a heated lid to minimize evaporation. At the end of the incubation time, reactions were quenched with 2 µL of 10 mM phenylmethyl sulfonyl fluoride, which rapidly and irreversibly inactivates the protease. The extent of proteolysis was measured by SDS-PAGE. Loading buffer was added to each sample to a final concentration of 2% SDS, 10% glycerol, and 0.1% 2-mercaptoethanol, and the samples were incubated at 98 °C for 6 min. Samples were run on 16% tris-glycine polyacrylamide gels (Thermo Fisher), stained with GelCode Blue (Thermo Fisher), and visualized using an ImageQuant LAS 4000 gel imaging system (Cytiva). Bands were quantitated with ImageLab software (Bio-Rad, Hercules, CA, USA). Remaining LC was defined as the intensity of the full-length LC band after proteolysis, relative to the un-proteolyzed LC sample, calculated independently for each of three gels.