Pmi phosphorimager

Binding of CsrA to RNAs was determined by EMSA with recombinant CsrA-His₆, purified as described previously (20 (link)), and RNA synthesized in vitro with MEGAshortscript Kit (Ambion). The template DNA for in vitro transcription of sRNAs was generated by PCR from MG1655 genomic DNA, using primers hflK Fwd T7 and hflK Rev T7. In vitro-transcribed sRNAs were gel-purified, treated with Antarctic phosphatase (NEB), and radiolabeled at the 5′ end using [γ-³²P] ATP and T4 polynucleotide kinase. Binding reactions contained 0.5 nM labeled RNA, 10 mM MgCl2, 100 mM KCl, 32.5 ng total yeast RNA, 20 mM DTT, 7.5% glycerol, 4 U SUPERasin (Ambion), and various concentrations of CsrA (0–400 nM) and incubated at 37 °C for 30 min. Reaction mixtures were separated on native polyacrylamide gels using 1X TBE as the electrophoresis buffer, gels dried and imaged using a PMI phosphorimager (Bio-Rad). The signals of free (F) and shifted/bound RNA-Protein (B) complex were quantified with the help of Quantity One software (Bio-Rad). A linear regression analysis of the data was performed to calculate the apparent equilibrium binding constant (Kd) for the RNA-protein interaction (Fig. S5). Data presented in the Fig. S5 are averages of two independent experiments.

Binding of CsrA to RNAs was determined by EMSA with recombinant CsrA-His₆^{17 (link)} and RNA synthesized in vitro with MEGAshortscript Kit (Ambion) or the Ampliscribe T7 flash Transcription Kit (EpiCentre). The template DNA for in vitro transcription of sRNAs was generated by PCR from MG1655 genomic DNA, using the primers listed in Supplementary Data 10. In vitro-transcribed sRNAs were gel-purified, treated with Antarctic phosphatase (NEB), and radiolabeled at the 5′ end using [γ-³²P] ATP and T4 polynucleotide kinase. Binding reactions contained 0.1–0.5 nM RNA, 10 mM MgCl₂, 100 mM KCl, 32.5 ng total yeast RNA, 20 mM DTT, 7.5% glycerol, 4 U SUPERasin (Ambion), and various concentrations of CsrA (0–400 nM), and incubated at 37 °C for 30 min. Reactions mixtures were separated on native polyacrylamide gels using 1× TBE as the electrophoresis buffer and labeled RNA was analyzed using a PMI phosphorimager (Bio-Rad) and Quantity One software (Bio-Rad), as described previously^{17 (link)}. Apparent equilibrium binding constants (K_d) presented in the figures are averages of two independent experiments.

Peptides 19 amino acids long were synthesized on cellulose membranes using SPOT technology^{48 (link)}. Membranes were rinsed with 95% ethanol, followed by washing 5× with TBST. GSK3 kinase (NEB) was then prepared in a reaction mixture comprising 50 ng of kinase, 200 μM ATP (Sigma-Aldrich) and 8 µCi [γ-³²P]-ATP (Perkin Elmer) in 1.5 ml of 1× NEBuffer for Protein Kinases (NEB). The reaction mixture was then placed on the membrane, which was incubated at 30 °C for 30 min. The membrane was then washed 15× with NaCl (1 M), followed by washing 10× with water before washing with stripping buffer (1% SDS, 8 M urea, 0.5% 2-mercaptoethanol) for 1 h at 40 °C. This was followed by washing 10× in water and a final wash in 95% ethanol. After air drying the membrane for 10 min, it was exposed to a phosphor screen overnight for 3 h. Images were acquired with a PMI phosphorimager (Bio-Rad).