pRb is also involved in NET4/Tmem53 effects.

<p>(A) pRb was knocked down by siRNA in HEK293T cells subsequently transfected with NETs. Cell cycle profiles are shown for these cells expressing NET4/Tmem53 or controls of mRFP alone or NET51/C14orf1 that had no effect in the original flow cytometry screen. The 2N accumulation effect of NET4/Tmem53 on the cell cycle profile observed in HEK293T cells was lost when pRb levels were reduced. The knockdown of pRb is confirmed in the lower left corner. (B) Photomicrographs of cells overexpressing the RFP alone control or NET4/Tmem53. Cells were stained with an antibody that recognizes pRb phosphorylated on serine 780. NET/mRFP signal is shown in the left panels to identify transfected cells and phospho-pRb staining in the right panels. In each panel two adjacent cells are marked by arrowheads, one transfected and the other not transfected. All micrographs were taken at the same exposure time. The graph on the right shows quantification of the pixel intensity for the phospho-pRb staining. The pixel intensity for untransfected controls internal to each micrograph was set to 1 and average relative values from 40 transfected cells and 40 untransfected cells for each transfection are shown with standard errors.