Hrp labelled secondary antibody

The following antibodies, beads, and dyes were obtained commercially: HCV core (clone 7–50, sc-57800), HA (clone Y-11, sc-805-G), PABPC1 (clone 10E10, sc-32318), MOV10 (clone B-3, sc-515722), calnexin (c-20, sc-6465), TIA1 (c-20, sc-1751) (all Santa Cruz Biotechnology), G3BP1 (Clone 23/G3BP, 611127, BD Biosciences), PLIN2 (GP40, Progen; ab52356, abcam), HCV NS3 (ab65407, abcam), HCV NS5A (HCM-131-5, IBT), FLAG (F7425), FLAG (F1804), HA (H6908), tubulin (clone B5-1-2, T6074), anti-FLAG M2 affinity gel (A2220), anti-HA affinity gel (HA-7, A2095), recombinant protein A (15918–014) and protein G (15920–010) agarose beads (all Sigma), L1ORF1p (clone 4H1, MABC1152, Merck), YB-1 (ab12148, abcam), HRP-labelled secondary antibodies (Jackson ImmunoResearch), HRP-labelled TrueBlot secondary antibodies (Rockland Immunochemicals), Alexa488-, Alexa555-, and Alexa647-conjugated secondary antibodies (all donkey, IgG (H+L)), BODIPY493/503 (D-3922), BODIPY 655/676 (B-3932) (all Life Technologies), Hoechst33342 (Thermo Fisher). L1ORF1p antibody #984 was described previously [33 (link)]. Oligonucleotides and PCR primer were purchased from Sigma. Restriction enzymes for molecular cloning were obtained from NEB, other enzymes from Thermo Fisher. Unless stated otherwise, chemicals were purchased from Sigma or Applichem and cell culture reagents from Gibco/Thermo Fisher.

Samples were homogenized in M-PER® Mammalian Protein Extraction Reagent lysis buffer (Thermo, MI, USA) containing protease inhibitor cocktail and 0.5 M EDTA (Thermo, MI, USA) (1:100 dilution). The supernatant was collected after centrifugation at 12000 rpm for 30 min at 4 °C. The protein concentration was determined using the BCA Protein Assay Kit (Thermo, MI, USA). The protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 h and then incubated with anti-TTF1 (1:500, Abcam, MA, USA), anti-Kiss1 (1:400, Abcam, MA, USA) or anti-β-tubulin antibody (1:1000, CST, MA, USA) at 4 °C overnight. Subsequently, the membranes were rinsed three times with Tris-buffered saline with 0.1% Tween 20 (TBST) every 10 min and probed with an HRP-labelled secondary antibody (1:10000, Jackson, PA, USA) at room temperature for 1 h. After three additional rinses with TBST, the membranes were visualized using the ECL system. The grey values of the protein bands were analysed using ImageJ software.

Dot blot assay was used to analyze genomic DNA that was isolated from P. torridus cultures as described above. For this, 1 μg genomic DNA was digested with EcoRI and then denatured with 0.3 N sodium hydroxide for 30 min. This was followed by neutralization with 1 M ammonium acetate before spotting on Hybond N⁺ nylon membrane (GE Healthcare, United States) using the Bio-Dot apparatus (Bio-Rad Laboratories, United States). The membrane was baked at 80°C for 2 h before probing with anti-m6A antibody (Sigma Aldrich, Cat. No. ABE572) or anti-m5C antibody (EpiGentek, Cat.No. A-1014-010). This was done by blocking the baked dot blots with 10% skim milk (Difco Laboratories) for an hour at room temperature, washing the blots twice with 1X PBS-T, and then incubating them overnight at 4°C with the anti-m6A or anti-m5C antibodies (1:1000 dil in 1X PBS). Following three washes with 1X PBS-T the blots were incubated with HRP-labelled secondary antibody (1:10000 dil, Jackson Laboratory, United States) for an hour and developed using a chemiluminescence method.