To determine the effect of inS3-54 on STAT3 or STAT1 binding to SIE probe, inS3-54 was first diluted with DMSO, and equal volume of diluted inS3-54 waddedded to the reaction mixture followed by incubation at RT for 30 min before incubating with the labeled SIE probe.
For HDV binding inhibition assay, peptides were pre-incubated with target cells at 16°C for 1 hr before incubating with the virus; antibodies against viral envelope protein were pre-incubated with viruses before adding to target cells.
The dried membrane was washed 5 6 times with methanol (to remove residual Coomassie Blue G-250), rinsed with water and then blocked for 2 h at room temperature in 5% milk, before incubating with the desired antibodies diluted in 2.5% milk overnight at 4 °C.
The cells were re-suspended in Annexin-V binding buffer (BD, Oxford, UK) before incubating with monoclonal antibody Annexin-V conjugated with Alexa 488 (10 μg/ml) (Sigma, UK) for 30 min avoiding light.
with 0.1% Tween-20/PBS Tween-20/PBSating with primary antibefore.
For all experiments except those for Rap1, cells were plated 24 h before incubating with ESCAs or metabolites.
Plates were blocked with BSA and washed twice with PBST before incubating with the biotinylated peptides (50 ng/well) at room temperature for 1 hour.
The primary antibody was removed and samples washed again in blocking solution three times before incubating with appropriate secondary antibodies conjugated to specific fluorophores for 1 hour at room temperature.
Fractionated proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore) and membranes blocked for 1 hour at room temperature with 5% skim milk/TBST, before incubating with primary antibody overnight at 4°C.
The membranes were blocked in 5% BSA before incubating with the following monoclonal antibodies: HIF-1α (Novus Biologicals), pAkt1 (Cell Signaling Technology), Akt1 (Cell Signaling Technology), pMAPK (Cell Signaling Technology, MAPK Cell Signaling Technologygy), or total actin (Sigma).
For Rap1 assay, cells were plated 48 h before incubating with 8CPT-2'-OMe-cAMP or Sp-8CPT-2'-OMe-cAMP. Media from experiments was either assayed immediately after collection or frozen (−20°C) until all samples were available.
