HSF1-interacting proteins in control and heat shock conditions

HeLa cells were treated or untreated with heat shock at 42°C for 5, 30, and 60 min, and were then fixed in medium containing 1% formaldehyde at 37°C for 30 min. Chromatin fraction was sonicated with the Branson Sonifier 450 (BRANSON Ultrasonics) into fragmented DNA around 200-300 bp. HSF1 bound to genomic DNA was immunoprecipitated with 2 ul of rabbit immune serum for mHSF1 (anti-mHSF1j or Millipore ABE1044) or preimmune serum conjugated to Dynabeads Protein A at 4°C for 3 h (ChIP). Immunoprecipitated sample was eluted by incubation at 90°C for 30 min in 1× Laemmli sample buffer (2% SDS, 60 mM Tris-HCl (pH 6.8), 100 mM DTT, 10% glycerol, 0.001% bromophenol blue). Protein samples were loaded on 12% SDS-PAGE for preparation of gel slices for mass spectrometry (MS). Gel bands were cut-out and subjected to in-gel digestion with trypsin. Resulting peptides were dissolved in a solution containing 0.1% trifluoroacetic acid and 2% acetonitrile and analyzed by an LTQ Orbitrap Velos Pro mass spectrometer coupled with a nanoLC instrument and HTC-PAL autosampler.