QUICK SEARCH:

[advanced]

	Author:		Keyword(s):

Home Help Feedback Subscriptions Archive Search Table of Contents

	Help viewing high resolution images
	Return to article

(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.

Fig. 1. Specific depletion of 23C ${gamma}$ -tubulin by RNA-mediated interference (RNAi). (A) Depletion kinetics. Control cells (C) and cells subjected to 23C ${gamma}$ -tubulin RNAi treatment ( ${gamma}$ 23C) were harvested after 5 days, 7 days and 9 days of culture. Total-protein extracts (50 µg) were analysed by immunoblotting. From day 5 to day 9, a strong and specific depletion of 23C ${gamma}$ -tubulin was observed. (First and second rows) Monoclonal GTU ${gamma}$ -tubulin antibody staining. The 37CD ${gamma}$ -tubulin band was not visible when the signal from 23C ${gamma}$ -tubulin was not overexposed (first row) and could be visualized only by reacting blots with more antibody and then overexposing the membranes (second row). (Third row) Purified polyclonal R70 antibody staining. Here, 37CD ${gamma}$ -tubulin also appeared to be less abundant than the 23C ${gamma}$ -tubulin. (Fourth row) The monoclonal antibody mab1501 was used to detect actin as an internal loading control. (B) Quantification of the depletion of ${gamma}$ -tubulin. The ${gamma}$ -tubulin content of 40 µg of an extract from cells submitted to a 7-day RNAi ( ${gamma}$ 23C) was compared by immunoblotting with that of serial loadings (20-1.25 µg) of protein extracts from control cells (C) and quantified by densitometry analysis. 23C ${gamma}$ -tubulin RNAi reduced the ${gamma}$ -tubulin level by at least 95%. (C) Reversibility of ${gamma}$ -tubulin depletion. Extracts (50 µg), prepared from control cells (C), 7-day-treated cells ( ${gamma}$ 23C) or from cells that had been treated with the ${gamma}$ 23C RNA for 7 days, then washed and maintained in culture for a further 7 days (R7) and 14 days (R14) without RNAi were analysed by immunoblotting with the GTU (against 23C ${gamma}$ -tubulin) and mab1501 (against actin) antibodies. The 23C ${gamma}$ -tubulin signal reappeared 14 days after the removal of the ${gamma}$ 23C RNA by washing. (D) Depletion specificity. Extracts (50 µg) from cells submitted to a 7-day RNAi treatment against either 23C ${gamma}$ -tubulin ( ${gamma}$ 23C) or the transcriptional factor Cdk8 (cdk8) were immunoblotted with the antibodies GTU (against 23C ${gamma}$ -tubulin) and mab1501 (against actin). Depletion of Cdk8 did not affect the levels of 23C ${gamma}$ -tubulin and actin.

Return to article