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220 Zhang et al.Fig. 5. Replacement of endogenous ASF1a by ectopically expressed HA-taggedASF1a. W138 cells were infected with mock (1,4) virus encoding shRNA to ASF1a(2,5), or virus encoding shRNA to ASF1a and a myc-tagged shRNA-resistant form ofASF1a (3,6). Cell extracts were Western blotted with anti-ASF1a or anti-myc antibodies.13. Remove the medium from the WI38 cells 24 h postinfection and replace with 10 mLof fresh WI38 medium, containing puromycin at a final concentration of 3 µg/mL.14. Typically, 3 d after addition of puromycin, all of the noninfected cells should bedead. At this time, there should be no surviving cells left on the mock virusinfected plate.15. Harvest the infected cells for Western blot analysis by scraping directly in 1XLaemmli sample buffer, followed by boiling for 4 min. Samples can be storedfrozen at −80°C.3.5. Testing Protein Knockdown and Ectopic ExpressionCell extracts should be fractionated by SDS-PAGE, transferred to a PVDFmembrane and then Western blotted with antibodies to the protein of interest andwith anti-HA antibodies (or an appropriate epitope tag) (10). During SDS-PAGE,it should be possible to resolve the HA-tagged ectopically expressed protein fromthe untagged endogenous protein, making it possible to detect knockdown of theendogenous protein and its replacement by the ectopically expressed HA-taggedprotein of higher molecular weight. This is illustrated for ASF1a in Fig. 5.Once knockdown of the endogenous protein and expression of the ectopicprotein are confirmed, the cells can be assayed for the functional consequences.The assays herein will obviously be specific to each protein and researcher.However, regardless of the assay, the following control viruses should also beused: a virus that knocks down the endogenous protein, but does not directexpression of an ectopic protein; a virus that knocks down the endogenousprotein, and directs expression of the HA-tagged wild-type protein.4. Notes1. About 1 µg/mL Puromycin should kill all of the untransfected cells within 48 h.The efficiency of transfection and cell killing should be determined from the plates