. Author manuscript; available in PMC: 2012 Dec 1.

Published in final edited form as: Cytoskeleton (Hoboken). 2011 Nov 29;68(12):671–693. doi: 10.1002/cm.20544

Table 1.

Measurement of F-actin binding to CD44-associated AFAP-110 complex isolated from MDA-MB-231 cells.

(A) Effects of anti-CD44 antibody on HA-dependent ¹²⁵I-F-actin binding to the CD44-AFAP-110 complex:
Treatments	¹²⁵I-F-actin binding to the CD44-AFAP-110 complex (cpm) (% of control)
No treatment (control)	12,742 ± 637 (100%)
LMW-HA treatment	38,353 ± 3,068 (300%)^a
Anti-CD44 IgG + LMW-HA treatment	12,487 ± 250 (98%)^a
HMW-HA treatment	13,761 ± 550 (108%)^a
HA fragment (2–3 disaccharide) treatment	11,722 ± 423 (92%)^a
Normal IgG + no HA treatment	12,500 ± 573 (98%)^a
Normal IgG + LMW-HA treatment	37,981 ± 2,550 (298%)^a

(B) Effect of AFAP-110 siRNA on HA-dependent ¹²⁵I-F-actin binding to the CD44-AFAP-110 complex:
Treatments	¹²⁵I-F-actin binding to the CD44-AFAP-110 complex (cpm) (% of control)
Treatments	Scrambled siRNA-treated cells	AFAP-110 siRNA treated cells
No treatment (control)	10,980 ± 439 (100%)	9,309 ± 186 (85%)^b
LMW-HA treatment	31,842 ± 2,865 (290%)^b	9,529 ± 381 (87%)^b
HMW-HA treatment	11,529 ± 576 (105%)^b	9,200 ± 276 (84%)^b
HA fragment (2–3 disaccharide) treatment	10,138 ± 423 (92%)^b	9,187 ± 154 (84%)^b

The procedures for measuring ¹²⁵I-F-actin binding to the CD44-AFAP-110 complex using MDA-MB-231 cells [treated with 1µg/ml LMW-HA or HMW-HA or HA fragment (2–3 disaccharide) (or pretreated with rat anti-CD44 IgG followed by adding 1µg/ml LMW-HA or no HA) (or normal IgG alone or normal IgG plus LMW-HA) or transfected with scrambled siRNA or AFAP-110 siRNA in the presence or absence of 1µg/ml LMW-HA or HMW-HA] are described in the Materials and Methods. The amount of F-actin binding to the CD44-associated AFAP-110 complex isolated from untreated cells (Table 1A-control) or MDA-MB-231 cells treated with scrambled siRNA without HA (Table 1B-control) is designated as 100%. All data represent mean ± SEM of the amount of ¹²⁵I-F-actin binding to the CD44-AFAP-110 complex detected in each sample. The value represents an average of triplicate determinations of six experiments with an S.D. value less than ±5%.

Statistically significant (p<0.001; analysis of variance; n=6) as compared with control samples [e.g., untreated cells (control)].

Statistically significant (p<0.005; analysis of variance; n=6) as compared with control samples [e.g., scrambled siRNA-treated cells (control) without HA].