Microtubule movements on the arms of mitotic chromosomes: Polar ejection forces quantified in vitro

Brouhard and Hunt. 10.1073/pnas.0506017102.

Supporting Information

Files in this Data Supplement:

Supporting Text
Supporting Figure 4
Supporting Figure 5
Supporting Figure 6
Supporting Table 1
Supporting Table 2

Supporting Figure 4

Fig. 4. Quantitative fluorescence calibration. The diagram shows the truncated-pyramid- shaped volume extending from the coverglass to the microscope slide. The colored slice is an individual integral plane, dz.

Supporting Figure 5

Fig. 5. Calibration data for quantitative fluorescence measurements.

Supporting Figure 6

Fig. 6. Examples of fluorescent (anti-Kid) chromosome images used to quantify motor density. The control is stained with DAPI only.

Table. 1. Fluorescence intensity values measured for chromosomes

Sample	n	I/pixel	I/mm²	Motors/mm²
DAPI-only control	10	2 ± 4	5,184 ± 10,368	Not applicable
Anti-Kid	25	123 ± 31	318,816 ± 80.352	400 ± 100
Anti-KIF4	14	94 ± 15	243,648 ± 38,880	300 ± 50

Table 2. Antibody inhibition of MT binding

[MT]	Sample	n	No. of MTs per chromosome (e_B)
1:10	DAPI	5	11.2 ± 0.8
	Anti-Kid	5	2.4 ± 1.1
	Anti-KIF4	5	7.2 ± 0.8
	Anti-topoIIa	5	10.0 ± 1.6
1:100	DAPI	15	1.6 ± 0.7
	Anti-Kid	11	0.0 ± 0.0
	Anti-KIF4	5	0.8 ± 0.4
	Anti-topoIIa	5	1.8 ± 0.4
1:1000	DAPI	5	0.6 ± 0.5
	Anti-Kid	5	0.0 ± 0.0
	Anti-KIF4	5	0.0 ± 0.0
	Anti-topoIIa	5	0.4 ± 0.5

The table lists the complete results from the antibody-binding experiments performed using antibodies to the chromokinesins Kid and KIF4. The MT concentration ([MT]) is stated as the dilution factor for a polymerized aliquot of 5 mg/ml tubulin dimer. topoIIa, Topoisomerase IIa.