Fig. 3
From: HPV 16E7 and 48E7 proteins use different mechanisms to target p130 to overcome cell cycle block
The LXCXE motif of HPV16 E7 binds to p130. a 20 μg of each pMSCVpuro-16E7-HA was calcium phosphate co-transfected with each type of p130 mutants for transient expression in T98G cells. In addition, the pMSCVpuro vector was transfected in T98G cells as a control. Transfected cells were puromycin selected and nuclear lysates were harvested 48 hours post transfection. Nuclear lysates were immunoprecipitated with HA (Roche) antibody, separated on a 15 % SDS-PAGE gel and western blotted onto a PVDF membrane. 16E7 proteins were detected using HA (Roche) antibody. The image is representative of three independent experiments. b Nuclear lysates from T98G cells transfected with pMSCV puro constructed with 16E7HA or p130 wt, p130 mE7, p130PM2, p130mE7/PM22 and a combination of 16E7 with each of the p130s. Transfected cells were puromycin selected and nuclear lysates were harvested 48 hours post transfection. Nuclear lysates were separated on a 10 % SDS-PAGE gel and western blotted onto a nitrocellulose membrane. p130 proteins were detected using HA (Roche) antibody. The image is representative of three independent experiments. c Detection of ectopically expressed p130 in immunoprecipitates of T98G cells transfected with pMSCV puro constructed with p130 wt, p130mE7, p130PM22, p130mE7/PM22 and a combination of 16E7 with each of the p130 constructs. Pre-immune serum (PI) and Lin-9 antibodies were used for immunoprecipitation and HA p130 was detected on western blots. The co-immunoprecipitation control, Lin-9 was detected using Lin-9 (Abcam) antibody. The image is representative of three independent experiments. (d) Flow cytometry of propidium iodide-stained T98G cells transfected with pMSCV puro constructed with p130wt, p130mE7, p130PM22, p130mE7/PM22 together with 16E7HA. The T98G cells transfected with pMSCV puro only was used as a control. The estimated percentages of cells in G1, S, and G2/M phase are shown