Table 2.

Quantitation of the steady-state levels of M6PR in isolated TR- and EGFR-containing endosomes

Time (min)	EGFR % PNS	TR % PNS	M6PR % PNS	% TR/ % EGFR	% M6PR/ % EGFR
10	18.1 ± 0.3	13.1 ± 0.4	3.5 ± 0.1	0.72	0.19
25	24.8 ± 0.1	5.3 ± 0.8	3.0 ± 0.2	0.22	0.12
45	11.2 ± 0.5	2.9 ± 1.1	2.0 ± 1.2	0.24	0.17
60	9.1 ± 0.7	2.4 ± 0.8	1.6 ± 0.2	0.25	0.17
No gold	0.1	NDa	0.2	–	–

Radiolabeled HEp-2 cells were preincubated with anti-EGFR-gold for 30 min at 37°C and then stimulated with saturating EGF (200 ng/ml) for up to 60 min at 37°C. Cells were then lysed and fractionated on sucrose density gradients as described in MATERIALS AND METHODS. The amounts of EGFR and TR in the isolated fractions were determined by Western blotting and for M6PR by immunoprecipitation. In the right hand panels, to correct for the differences in endosome isolation, the yields of TR and M6PR have been expressed as a proportion of the EGFR yield. When no anti-EGFR-gold conjugate is added to the fractionation procedure, ≤0.2% of M6PR or TR is recovered. Note that the yield of M6PR recovered in endosome fractions is low at all time points. 

^a ND, not detectable.