Table 1.

Extraction conditions withstood by strings in live cells at 4°C

No.	Extraction	Rationale of use
1	100 mM NaOAc, pH 5.5, 5 min	Disrupt pH-dependent interactions
2	100 mM Na2CO3, pH 11, 3 min	Disrupt pH-dependent interactions
3	10 µg/ml PPS, 30 min, or 500 µg/ml PPS, 1 h, or 1 µg/ml HM, 90 min (in PBS)	Compete with interactions with sulfated glycosaminoglycans
4	10 µg/ml PPS in 100 mM Na2CO3, 3 min	Combines conditions 2 and 3
5	2 M NaCl, 2 min	Disrupts electrostatic interactions including interactions with GAGs
6	0.1% Triton X-100 in PBS, 5 min	Does not extract GPI-APs (Mayor and Maxfield, 1995)

(Nos. 1–5) Live ScGT1 cells on Permanox slides were chilled to ice temperature, treated as detailed, and then fixed and stained with 8B4, except for HM-treated cells, which were immunostained on ice before fixation. (No. 6) For the Triton X-100 treatment, cells chilled to 4°C were first immunostained, then incubated with Triton X-100, and then fixed.