G3 245

Three-micrometer sections of formalin fixed and paraffin embedded tumor tissues were stained as previously described [11 (link)] with an antibody targeting RB1 (G3-245; BD Pharmingen).

C33A cells were transfected with either HA-E2F1-3 (along with DP1), myc tagged CDH1 or pRB expression plasmids under the control of CMV promoters using standard calcium phosphate precipitation techniques. 40 h after transfection cells were washed and collected in GSE buffer (20 mM Tris, pH 7.5, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol, 5 µg/mL leupeptin, 5 µg/mL aprotinin, 0.1 mM Na₃VO₄, 0.5 mM NaF, and 1 mM DTT) and frozen at −80 °C. Cell extracts were centrifuged and the supernatant was diluted twofold in low salt GSE (20 mM Tris, pH 7.5, 1.5 mM MgCl₂, 0.2 mM EDTA, 25 mM DTT, 0.1% NP-40) and combined with glutathione beads and recombinant fusion proteins. GST-RB large pocket (amino acids 379–928) and GST-HPV-E7 recombinant proteins were expressed and purified as previously described [29 (link)]. Beads were then washed twice with low salt GSE, boiled in SDS-sample buffer, resolved by SDS-PAGE, and western blotted. HA-tagged proteins were detected using anti-HA 3F10 (Roche), myc-tagged CDH1 was detected using monoclonal antibody 9E11, and pRB was detected with G3-245 (BD Pharmagen). In order to test pRB stability, cells transfected with CMV expressed pRB were treated with 100 µg/mL cycloheximide for 24 h. Extracts were prepared in GSE buffer every 3 h up to 15 h. Extracts were spun down and western blotted for pRB.