20s proteasome

Plasmid cDNA of HIF-1α was transfected to SY5Y cells. Cell extracts prepared in modified M2 buffer was incubated with anti-HIF-1α antibody and protein A-sepharose beads (Pharmacia) at 4°C overnight. The beads were precipitated and washed five times with M2 buffer. One tenth of the beads were used to verify the bound HIF-1α protein. Western blot for the proteasome 20S subunits (antibodies: Santa Cruz, 1:1,000) was included to confirm no contamination of the proteasomes. The protein on beads was incubated with H₂O₂ (0.03%) and Fe²⁺ (0.1 mM) in DMEM for 3 h. Precipitated HIF-1α protein with or without oxidation by H₂O₂ were incubated with 20S proteasome (Boston Biochem) to determine the proteasome's ability to degrade HIF-1α. To determine the ability of 26S proteasome, the precipitated HIF-1α was incubated with a cytosol fraction from SH-SY5Y cells and 26S proteasome (Boston Biochem). After HIF-1α was incubated with the proteasomes for 1 and 3 h, Western blotting was carried out to determine the level of HIF-1α. MG-132 was used to confirm that the degradation was in fact the result of proteasome activity.

We analyzed the degradation kinetics of 200 ng/condition of recombinant H1.0, its GD and CTD, and 1 μg/condition of the perchloric acid purified histone mixture from T47D cells, using the protocol described by Kudriaeva et al. (2019 (link)). Bovine serum albumin (Sigma) was used as a negative control. Proteolytic digestion was performed in 20 mM Tris pH 7,5, 20 mM NaCl, 1 mM EDTA, and 1 mM DTT, in the presence of 5 nM of 20S proteasome (Boston Biochem, E‐360), at 37°C. The reaction was stopped by adding electrophoresis loading buffer and incubating the samples at 95°C for 5 min. The digestion products were analyzed by 12%–15% SDS‐PAGE and stained with Coomassie blue or silver (Silver stain plus kit, Bio‐rad). Images were taken using a Chemidoc imaging system (Bio‐Rad). Degradation intermediates were characterized by top‐down mass spectrometry.