Detection of 3xFlag7Q- and 3xFlag20Q-htt in the SDS-insoluble nuclear fractions from Hdh140Q/3xFlag7Qand Hdh140Q/3xFlag20Qbrains. (A) Aliquots of the crude nuclear fraction obtained from the whole brain of a 24 month-old Hdh 140Q/3xFlag7Q mouse were heated in the presence of SDS and analyzed by cellulose filter trap assay and western blotting. SDS-resistant htt inclusions trapped on the cellulose acetate membrane were detected with 1C2 and MAB5374 antibodies, while sequestered 3xFlag7Q-htt was detected with the FLAG M2 antibody. SDS-soluble 140Q-htt and 3xFlag7Q-htt species were detected on the PVDF membrane using 1C2 and FLAG antibodies. (B) Crude nuclear fractions were obtained 24 month-old Hdh 140Q/3xFlag7Q (140Q/3xF7Q), Hdh140Q/3xFlag20Q (140Q/3xF20Q), and Hdh 3xFlag140Q/HA7Q (3xF140Q/HA7Q) brains, boiled in the presence of SDS, sonicated, and the remaining SDS-insoluble material was extracted with formic acid. The formic acid-solubilized fractions were analyzed by western blotting using Flag (FLAG M2, left panel), and 1C2 antibodies (right panel). 3xFlag epitope immunoreactivity in the Hdh140Q/3xFlag20Q nuclear fraction is increased in comparison to the Hdh 140Q/3xFlag7Q fraction. The sizes of protein standards (in kD) are indicated.