lipofectamine 2000 mediated transfection  (Addgene inc)

Journal: PLoS ONE

Article Title: GNL3L Is a Nucleo-Cytoplasmic Shuttling Protein: Role in Cell Cycle Regulation

doi: 10.1371/journal.pone.0135845

Figure Lengend Snippet: ( A ) Schematic representation of wild type, nuclear import (GNL3L ΔNLS ) and nuclear export (GNL3L ΔNES ) defective mutants of GNL3L. ( B ) HeLa cells were transfected with Flag-tagged GNL3L WT , GNL3L ΔNES and GNL3L ΔNLS expression plasmids and the subcellular localization was analyzed using confocal microscopy. The scale bar represents 20μm. ( C ) HEK293 cells were transfected with Flag-tagged GNL3L WT , GNL3L ΔNES and GNL3L ΔNLS and the expression was determined by western blot analysis using anti-Flag antibody. (D) Ectopic expression of GNL3L results in decreased accumulation of ‘G2/M’ population. GNL3L WT was overexpressed in HEK293 cells and the asynchronous cell cycle pattern was analyzed using flow cytometry as described in Materials and Methods. (E) Western blot was performed to analyze the protein levels of endogenous cyclins A2 and B1 upon GNL3L WT expression in HEK293 cells. Beta actin served as loading control. (F) GNL3L knockdown leads to increased accumulation of cells in G2/M phase of the cell cycle. Transient knockdown of GNL3L was performed in HEK293 cells using specific siRNA and the cell cycle profile was analyzed using flow cytometry. (NC: Negative control). (G) Endogenous cyclins A2 and B1 levels were analyzed using western blot upon GNL3L knockdown in HEK293.

Article Snippet: Transient knockdown of GNL3L was performed by lipofectamine 2000-mediated transfection of siRNA in HEK293 cells (L-015743-01-0005, ON-TARGET plus SMARTpool, Human GNL3L (54552), Thermo Fisher Scientific Inc., USA).

Techniques: Transfection, Expressing, Confocal Microscopy, Western Blot, Flow Cytometry, Control, Knockdown, Negative Control