For purification, one pellet was thawed and resuspended into 50 mM KH2PO4 (pH 7.9), 300 mM NaCl, 20 mM imidazole, one complete protease inhibitor cocktail tablet (Roche) and DNAse and RNAse both at a final concentration of 50 µg/mL.
In a 50 µL PCR reaction, 25 µL of SYBR® Green PCR Master Mix, 2 µL of forward (5'-GAGGAAGGIGIGGAIGACGT-3') and reverse primers (5'-AGICCCGIGAACGTATTCAC-3') (both at a final concentration of 0.2 µM), 19 µL of H2O and 2 µL of DNA samples were added.
One reaction amplified the RT coding region with the forward primer JR23 5'-TAATGACAGGCGACACC-3' and the reverse primer JR24 5'-TGTGCTGCCCAATTTAG-3', both at a final concentration of 0.2 μM.
Briefly, tumor samples were dissociated to form a single-cell suspension, which was plated on serum-free DMEM/F12 supplemented with B27, heparin (Stem Cell Technologies, Grenoble, France) and human recombinant EGF and FGF-2 (Sigma, St . Louis MO, USA), both at a final concentration of 20 ng/ml.
Equal loads of protein were added in 96-well plates and mixed with 50 mM of fluorogenic substrates: Ac-DEVD-AFC (Millipore, Billerica, MA, USA) for Caspase-3-like activity or Ac-VDVAD-AFC (ENZO Life Sciences, Farmingdale, NY, USA) for Caspase-2 activity, both at a final concentration of 50 μM in 96-well plates.
Replicate cultures of 2×106 PBMCs were cultured for 6 hours in the absence (unstimulated) or presence (stimulated) of a single pool of HIV Gag peptides (15-mers, overlapping by 11; NIH AIDS Research & Reference Reagent Program), which were identically matched to the Gag antigen expressed by both MVA vectors, at a final concentration of 2 µg/peptide/ml.
Both were used at a final concentration of 1 μg/ml.
Note that for silencing of Mcl-1, we have transfected both the siRNAs together at a final concentration of 60 nM each.
Both siRNAs were used at a final concentration of 80 nmol/l.
After 24 h, pertuzumab, trastuzumab, and a 1 : 1 combination of both antibodies were added at a final concentration of 20 μg ml−1.
For TMV-iAlk, 25 molar excess of propargylamine per coat protein was reacted using EDC coupling with 45 molar excess of both EDC and HOBt at a final concentration of 2 mg/mL TMV in HEPES buffer.
