Fig. 2.
ABPP of melanoma cell proteomes with the SAHA-BPyne probe. (A) Soluble proteomes from the melanoma lines MUM2B and MUM2C were incubated with 100 nM SAHA-BPyne probe in the presence or absence of excess SAHA (10 μM) as a competitor. Probe targets were detected by UV-irradiation, followed by click chemistry with a rhodamine-azide tag, SDS/PAGE analysis, and in-gel fluorescence scanning (fluorescent gel shown in grayscale). Multiple SAHA-sensitive targets were detected (arrows). These proteins were identified as HDACs 1 and 2 (60-kDa doublet) and MBD3 (38-kDa band). (B) Confirmation that SAHA-BPyne targets both HDACs (HDAC2) and HDAC-associated (CoREST, MTA2) proteins. Shown are Western blots of proteins enriched from melanoma proteomes by treatment with SAHA-BPyne (or SAHA-BPyne plus excess SAHA), click conjugation to biotin-azide, and enrichment on avidin beads.