Depletion of Nuclear Poly(A) Binding Protein PABPN1 Produces a Compensatory Response by Cytoplasmic PABP4 and PABP5 in Cultured Human Cells
Figure 1
Knockdown of PABPN1 in human cells.
HeLa and/or HEK293 cells were grown on 35 mm plates to approximately 30% confluence and transiently transfected with PABPN1-Si or PABPN1-UTR RNAi for indicated length of time. Non transfected (NT) control cells were also maintained in culture for the same duration as transfected cells. (A, B) Following transfection, cells were harvested at 45 h, 66 h and 80 h in Laemmli buffer. Whole cell extracts from Si, UTR, and NT HeLa (A) and HEK293 (B) cells were analyzed for PABPN1 protein by western blotting; GAPDH was used as the loading control. (C) Forty five hours after transfection, HeLa cells grown on coverslip were fixed with Para-formaldehyde and processed for immunostaining with PABPN1 specific antibody and counterstained with Texas-red-conjugated secondary antibody. Processed specimens were then examined with a confocal microscope. Images from two different sections of the slide are shown here. (D) Forty five hours and sixty six hours after transfection, RNA was extracted from HeLa cells using Trizol and reverse transcribed; cDNA thus made were used for PCR with primers specific to β-actin, PABP and PABPN1. Samples from PABPN1-UTR Si and non transfected (NT) cells were collected at 66 h time point. The bands on the gel representing the PCR products were scanned and quantified as described in Materials and Methods. The band intensities are shown in arbitrary unit.