Fla7000 imager

Following transfection as described above, RNA was extracted from Huh7 cells then processed according to standard Northern blotting procedures [28 (link), 29 ]. ³²P-labeled oligonucleotides, HBV Surface R and hGAPDH R, were used for detection of HBV RNA and GAPDH mRNA respectively. Bands were detected using a FLA-7000 Imager (FUJIFILM), and signal intensity then measured using densitometry with ImageJ software [30 (link)]. Relative HBV RNA concentrations were calculated as a ratio to GAPDH RNA values.

Proteins were expressed from constructs similar to those used in the yeast two-hybrid system with a hemagglutinin tag (pGADT7-DEST; VRN2, EMF2 and POT1a proteins) or a myc-tag (pGBKT7-DEST; TRB proteins) using a TNT Quick Coupled Transcription/Translation system (Promega) in 50 µl reaction volumes according to the manufacturer’s instructions. The VRN2, EMF2 or POT1a proteins were radioactively labelled using ³⁵S-Met. The co-immunoprecipitation procedure was performed as described by Schrumpfová et al. 2008 (link). Input, Unbound and Bound fractions were separated by 10% SDS – PAGE, and analyzed using a FLA7000 imager (Fuji-film).

[³²P]-AcP was produced using E. coli acetate kinase (Sigma) and [γ−³²P]-ATP [59 (link)]. To obtain [³²P]-AcP, 0.2 U of acetate kinase (Sigma Aldrich) and 200 μCi of γ-ATP (6000 Ci/mmol, Perkin Elmer) were buffered in 25 mM Tris-HCl (pH 7.4), 60 mM NaAc, 10 mM MgCl₂ at 25°C for 30 min. 4 μg of PhoP or its derived mutants were incubated with [³²P]-AcP at 30°C for half an hour. The reactions were stopped by adding Sample buffer. Samples were separated by SDS-PAGE gels, and gels were exposed 12 h on a phosphor screen (GE Healthcare). The signal was visualized with the Fujifilm FLA7000 imager.
Phosphorylation of PhoP by PhoQ_cyto were performed as follows: 1 μg of the PhoQ_cyto protein was phosphorylated with 10 μCi of [γ−³²P]-ATP in 50 μl of phosphorylation buffer (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.5 mg/ml BSA, 1 mM MgCl₂), for 20 min at 37°C. Phosphotransfer was performed by adding the autophosphorylated PhoQ_cyto to 2 μg of the WT PhoP or mutants in the same buffer. The reaction was carried out at 37°C for 1 h and stopped by adding Sample buffer.

[³²P]-AcP was synthesized with E. coli acetate kinase and [γ-³²P]-ATP [66 (link)]. The reaction mixture contained 60 mM NaAc, 25 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.5 U of acetate kinase (Sigma Aldrich) and 50 μCi of γATP (6000 Ci mmol^-1, Perkin Elmer). The reaction was incubated at 25°C for 30 min and the AcP was separated from the enzyme using a Microcon-10 microconcentrator (Millipore). Four micrograms of PhoP WT, PhoP K201Ac and PhoP D52A proteins were separately incubated with [³²P]-AcP for 30 min at 25°C. The reactions were stopped by addition of SDS sample buffer. Samples were subjected to SDS-PAGE, and gels were exposed overnight on a phosphor screen (GE Healthcare) followed by being visualized using the Fujifilm FLA7000 imager. Proteins were visualized by Coomassie brilliant blue staining.

The total RNA and northern blotting was performed as previously described (25 (link),26 (link)). Briefly, total RNA from an equal number of cells was resolved on a 1% agarose gel and transferred to a Nylon membrane using TV400-EBK Maxi Electro blotter (Severn Biotech). The blot was hybridized with the [³²P] 5′-end labelled oligonucleotides complementary to pre-rRNAs (listed in Supplementary Table S7) and exposed to phosphorimaging plates which were scanned on a FLA-7000 imager (Fuji).