, Western Blotting Samples were prepared with cleared cellular lysates in RIPA buffer containing protease/phosphatase inhibitor cocktail (Pierce) and normalized with a BCA assay kit. The lysates were diluted with 4X LDS loading buffer (Invitrogen) + 10X NuPAGE sample reducing agent, mixed via vortex, and heat denatured at 70 C for 5 min, J. Med. Chem, vol.37, pp.4-12, 1994.
, The protein bands were then transferred to a 0.2 nitrocellulose (Cell Signaling Technology) membrane at 1.25 A (constant) per gel-membrane sandwich for 12 min on a semi-dry Pierce Power Blotter with in Pierce 1-Step Transfer Buffer. The membranes were washed for 5 min in 1X TBS and blocked in 2% nonfat milk in 1X TBST for 1 h at 23 C. The blocking solution was removed, and the primary antibodies were diluted into in 5% BSA in TBST (see table below for typical dilutions), followed by incubation with the membranes overnight ($18 h) at 4 C. Following overnight incubation, primary antibodies were removed, and the blots were washed with 3 x 10 mL 1X TBST for 5 min/wash. The blots were then incubated with the appropriate rabbit or mouse secondary antibody (from Cell Signaling Technologies) in 5% nonfat milk in 1X TBST. The secondary antibody was then removed, and the blots were washed with 3 x 10 mL 1X TBST for 5 min/wash. The blots were then incubated with the appropriate developing reagent (Luminata Classico, Crescendo, or Forte Reagent (Millipore)) for 1 min and the chemiluminescent signal was visualized on an AlphaInnotech ChemiImager. Following visualization, the blots were washed with 1X TBS, stripped with Pierce PLUS Western Stripping Buffer, and re-washed with 1X TBS, Bis-Tris Gels (Invitrogen) and electrophoresis was carried out at constant voltage: 50 V for 30 min then 150 V for $90 min in either 1X MOPS or 1X MES Running Buffer with NuPAGE antioxidant (Invitrogen). The MW standard for visualization was the Biotinylated Protein Ladder Detection Pack (Cell Signaling Technology)
, Total RNA was purified with the Direct-zol RNA Miniprep Kit (Zymo Research), according to the manufacturer's protocol. Following isolation, DNase treatment was performed by incubating with 5U Turbo DNase (Ambion) for 30 min at 37 C. The RNAs were quantitated by Nanodrop and quality was assessed on either an Agilent 2100 Bioanalyzer or TapeStation 2200. All total RNA samples with RNA Integrity Numbers
, RNA-Seq libraries were prepared with the Illumina TruSeq RNA Library prep kit and analysis was carried out on 1 x 75 bp sequencing reads as previously described, 2012.
, The built atomic models were placed into the human 80S ribosome structure (PDB: 5LKS) at the vicinity of the respective predicted cross-linking sites. The models were oriented based on the surface complementarity between the ligands and possible binding area located proximally to the cross-linking sites. The possible interacting residues were inspected visually and identified using COOT. Nucleotides from the rRNA or amino acids of ribosomal proteins within a cut-off distance of 10 Å from the main residues that cross-link with the tetracycline ligands were considered as potential interacting residues. We thereby identified key residues, NanoString analysis was carried out at the Whitehead Institute Genome Technology Core with a custom codeset for the target genes of interest. Data were analyzed with the NanoString nSolver Analysis Software, p.0, 2010.
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, D4B8) Rabbit mAb, vol.1, issue.1, p.0
, XP Rabbit mAb, vol.1, issue.D7D3, p.500
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, 14C10) Rabbit mAb, vol.1, issue.1, p.0
, D4Z8V) Rabbit mAb, vol.1, issue.1, p.0
, D11A8) Rabbit mAb, vol.1, issue.1, p.0
, 13E5) Rabbit mAb, vol.1, issue.5, p.0
, 11H10) Rabbit mAb, vol.1, issue.5, p.0
, Cell Chemical Biology, vol.25, pp.1506-1518, 2018.