Subcellular
fractions were prepared using the Rapid, Efficient, And Practical
(REAP) method48 (link) with minor changes. Briefly,
HEL cells were counted, seeded, and treated as described above. After
24 h, the cells were transferred to clean tubes and pelleted by centrifugation.
The cells were washed with ice-cold PBS (Gibco 10010015), ice-cold
0.1 mg/mL heparin (Sigma-Aldrich H3149) in PBS, and then with ice-cold
PBS. Cellular membranes were lysed with ice-cold 0.1% (v/v) Nonidet
P 40 (Fluka 74385) in PBS. Samples were centrifuged at 1000g and 4 °C for 1 min, and supernatants were transferred
to clean tubes prior to storage at −80 °C (cytoplasmic
fractions). Nuclear pellets were washed with ice-cold 0.1% (v/v) Nonidet
P 40, followed by ice-cold PBS. The nuclei were lysed in Clarity OTX
Lysis-Loading Buffer v2.0 (Phenomenex AL0-8579). Nuclear lysates were
centrifuged at 13,000 rpm and 4 °C for 10 min, and supernatants
were transferred to clean tubes prior to storage at −80 °C
(nuclear fractions). All fractions were thawed on ice. Cytoplasmic
fractions were diluted 1:1 in Clarity OTX Lysis-Loading Buffer v2.0.
Then, diluted cytoplasmic fractions and nuclear fractions were diluted
1:75 in ultrapure water prior to analysis by CL-qPCR.
fractions were prepared using the Rapid, Efficient, And Practical
(REAP) method48 (link) with minor changes. Briefly,
HEL cells were counted, seeded, and treated as described above. After
24 h, the cells were transferred to clean tubes and pelleted by centrifugation.
The cells were washed with ice-cold PBS (Gibco 10010015), ice-cold
0.1 mg/mL heparin (Sigma-Aldrich H3149) in PBS, and then with ice-cold
PBS. Cellular membranes were lysed with ice-cold 0.1% (v/v) Nonidet
P 40 (Fluka 74385) in PBS. Samples were centrifuged at 1000g and 4 °C for 1 min, and supernatants were transferred
to clean tubes prior to storage at −80 °C (cytoplasmic
fractions). Nuclear pellets were washed with ice-cold 0.1% (v/v) Nonidet
P 40, followed by ice-cold PBS. The nuclei were lysed in Clarity OTX
Lysis-Loading Buffer v2.0 (Phenomenex AL0-8579). Nuclear lysates were
centrifuged at 13,000 rpm and 4 °C for 10 min, and supernatants
were transferred to clean tubes prior to storage at −80 °C
(nuclear fractions). All fractions were thawed on ice. Cytoplasmic
fractions were diluted 1:1 in Clarity OTX Lysis-Loading Buffer v2.0.
Then, diluted cytoplasmic fractions and nuclear fractions were diluted
1:75 in ultrapure water prior to analysis by CL-qPCR.
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