TABLE 2:

Effect of Dab2 on the size, number, and composition of CCSs.

Parameter	Control + pCGT	Dab2 shRNA + pCGT	Dab2 shRNA + T7-p96	Dab2 shRNA + T7-p96NPF 1-5*
Median diameter of CCSs (nm)	242	224	240	252
Number of CCSs	588 ± 41	521 ± 31	557 ± 63	569 ± 59
Relative level clathrin/CCS	1	0.91	1.03	1.04
Relative level Eps15/CCS	1	1.07	0.96	0.76***
Relative level AP2/CCS	1	1.74***	1.23	1.09

Control and Dab2-deficient HeLa cells reexpressing vector alone, T7-p96, or T7-p96 NPF1-5* and clathrin light chain (LCa)-EGFP were grown on collagen-coated coverslips and fixed, permeabilized, and stained with anti-Eps15 or anti-AP2 antibody. Clathrin structures on the ventral surface were characterized. No difference in the median size, number of CCSs, or clathrin content (area × mean intensity) was detected. However, the Eps15 content per clathrin structure was decreased in cells expressing the T7-p96 NPF1-5* mutant, and the AP2 content was increased in Dab2-deficient cells. The Eps15 content in clathrin structures was obtained by multiplying binary images of clathrin and Eps15 (to obtain area of “colocalized” Eps15), and superimposing onto the raw Eps15 image (to obtain intensity).

***p < 0.001 by the Mann–Whitney test. This test was used because the data follow a nonparametric distribution. Approximately 2000 structures were analyzed for each condition. ImageJ was used to obtain measurements.