Methylation-sensitive regulation of TMS1/ASC by the Ets factor, GA-binding protein-alpha - PubMed (original) (raw)

Purification of the HS2–55bp binding activity. A, the HS2–55bp binding activity was isolated from HeLa S3 cell nuclear extract by cation exchange chromatography and a two-step DNA affinity chromatography scheme. HeLa S3 nuclear extract (415 mg) was divided in half, and each half was independently bound to a SP-Sepharose cation exchange column in 50 m

m

NaCl binding buffer and step eluted in 50 m

m

, 100 m

m

, 250 m

m

, 500 m

m

, and 1

m

NaCl. Methylation-sensitive binding activity in the fractions was monitored by electrophoretic mobility shift assay (EMSA) using the HS-55bp probe in the presence of 100-fold molar excess of unlabeled HS2–55bp methylated at both CpG sites as a competitor. B, methylation-sensitive binding activity from each round of purification eluted with the 250 m

m

fractions. Representative EMSA analysis is shown. Methylation-sensitive complexes A and C are indicated. C, fractions containing peak activity from the cation exchange column were combined and negatively selected on a DNA affinity column containing the oligomerized, methylated HS2–55bp oligonucleotide. Flow-through from the methylated column was then bound to a DNA affinity column containing the oligomerized, unmethylated, HS2–55bp oligonucleotide, and eluted in 500 m

m

NaCl. Binding and elution from the unmethylated column were repeated twice, with a final step elution in NaCl. Fractions were analyzed by EMSA using the HS-55bp probe in the presence of 100-fold molar excess of unlabeled HS2–55bp methylated at both CpG sites as competitor. A representative analysis of the third round of purification is shown. Lanes 1, probe alone (Pr); lane 2, input (In); lane 3, flow-through (Ft); lane 4, 10 column volumes of 50 m

m

NaCl wash, 250 m

m

NaCl, and 500 m

m

NaCl eluted fractions. D, fractions 1–6 (250 m

m

NaCl) from the third round of affinity purification (see C) were trichloroacetic acid precipitated, separated on an SDS-10% polyacrylamide gel, and stained with Gel Code Blue (Pierce). Bands A–D (arrows) co-eluting with HS2 binding activity were excised and subject to MALDI MS/MS.