Data For Cyclic Immonium Ion of Lactyllysine Reveals Widespread Lactylation in the Human Proteome
Uncropped scans of blots and gels in Figures and Extended Data.
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Cells were lysed in RIPA lysis buffer (Beyotime, cat. no. P0013B) supplemented with protease inhibitor cocktail. Protein concentrations were then determined by the BCA assay. The lysates were diluted by 4×XT Sample Buffer (Bio-Rad, Hercules, USA. cat. no. 1610791), heated to 99 ˚C for 5 min, cooled and separated by 10% SDS-PAGE gel. After being transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, cat. no. 1620177), proteins were blocked using 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 detergent (TBST) and incubated with primary antibodies at 4 ℃ overnight. After being washed five times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 37 ˚C. The immunoblotted bands were detected by the addition of HRP substrate (Bio-Rad, cat. no. 170-5601) and captured on a ChemDoc XRS+ System (Bio-Rad). The primary antibodies used in this study include antibody against His-tag (Proteintech, cat. no. HRP-66005) at 1:5000 dilution and antibody against lactylation (PTM Bio Inc, cat. no. PTM-1401RM) at 1:2000 dilution for immunoblotting purified ALDOA, antibody against FLAG-tag (Cell Signaling Technology, cat. no. 14793S) at 1:1000 dilution for immunoblotting DHRS7, antibody against HDAC3 (Proteintech, cat. no. 10255-1-AP) at 1:1000 dilution, antibody against GAPDH (Abways Technology, cat. no. AB0037) at 1:3000 dilution, antibody against α-tubulin (Proteintech, cat. no. 11224-1-AP) at 1:2000 dilution.