New gene implicated in cancer, cellular stress response northwesternu NatureComms
. The settings were as follows, msa_mode: MMseqs2 , pair_mode: unpaired+paired, model_type: auto , and num_recycles: 3. Resultant HAPSTR2 PDB files were aligned with HAPSTR1 structures weighting the 80-152 region using the PyMOL2 command “new_structure AND resi 80-152, reference_structure”.Cell lines were obtained from ATCC and cultured according to the manufacturer’s guidelines. Cells were passaged with Accumax and tested regularly for Mycoplasma contamination.
For lentivirus, lentiviral vectors containing DNA constructs of interest were reverse co-transfected with pMD2.G and psPAX2 into 293T cells using Lipofectamine 3000 at the ratio recommended by the manufacturer. Media on transfected cells was changed 16 hours post-transfection, then the new virus-containing media was harvested 48 hours after initial transfection. Media was then centrifuged at 1000 ×and filtered with an 0.45 µm filter to yield packaged lentivirus.
Immunoprecipitations from mammalian cell lysates used anti-FLAG or anti-HA magnetic beads , which were pre-washed 3× with lysis buffer . Protein was then added to the beads before overnight incubation while rotating at 4 °C. Beads were washed 3× in lysis buffer prior to elution using glycine or being boiled in 2× SDS sample buffer.
Amylose pulldown experiments used 100 μl of amylose resin , which was pre-washed 5x with wash buffer before being incubated with either 750 μl of wash buffer, 750 μl of 5 μM purified His6-MBP, or His6-MBP-HAPSTR1 . Control beads and amylose-bound recombinant protein were washed 5x using wash buffer. 2.5 mg of protein from 293T whole cell lysates was then added to protein-immobilized beads and incubated overnight at 4 °C.
For our analyses, data in the Scaffold viewer were further filtered to a 1% false discovery rate for protein identification with a minimum of 2 peptides and 90% peptide threshold. Total spectra data were then exported and abundances were compared with the empty vector control IPs performed and analyzed in parallel. At least one sample needed >5 spectra to be included in our analysis. The average of two biological replicates is visualized for each condition.
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