Bolt lds sample buffer 4

WT and LH3 KO cells were plated and grown to 80 to 90% confluency. Following stimulation of procollagen biosynthesis with ascorbic acid for 1 day, the medium was replaced to the fresh standard DMEM, and the cells were cultured for 2 days. The medium containing secreted proteins was dissolved in Bolt LDS sample buffer 4× (Life Technologies) with reducing agents. After the cells were washed with PBS once, cell lysates were extracted using M-PER (Thermo Fisher Scientific) containing Halt Protease Inhibitor Cocktail, EDTA-Free (Thermo Fisher Scientific) at 4 °C according to the manufacturer’s instructions. After centrifugation, soluble proteins in the supernatant were mixed with the same sample buffer with reducing agents. These protein solutions were separated on SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes, and Western blots were performed. Gels, buffers, transfer condition, and antibodies are listed in Table S4. Blots were developed with horseradish peroxidase–enhanced Super-Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and detected by ChemiDoc MP imaging system (Bio-Rad) using the software Image Lab, version 4.0.1 (Bio-Rad). The intensities of protein signals were measured by ImageJ (National Institutes of Health).