Western blot and quantitative RT-qPCR (RT-qPCR) of marker proteins/transcripts for HEK293-subcellular compartments were used to verify the subcellular fractionation results. Western blots were performed with antibodies for three proteins – GAPDH (Abcam), SNRP70 (Abcam) and histone H3 (Abcam). Samples of subcellular fractions were boiled at 95°C for 10 minutes, then spun at 14000g for 3 minutes at room temperature to minimize the influence of sticky DNA (especially in the chromatin samples) on Western blots. For every Western blot, 1% of the sample volume was used. For RT-qPCR, the same percentage (1%) of RNA samples was used. Then the ratio of each marker gene (GAPDH, U1, ACTIN [intron]) was calculated in the respective chromatin, nucleoplasmic and cytoplasmic fractions.
For mES cells, similar western-blot experiments were carried out using antibodies against actin (Abcam), histone H3 (Abcam) and SNRP70 (Abcam). We confirmed NAI-N3 treatment didn’t affect fractionation. Protein was quantified using the Pierce BCA protein assay kit (Thermo Fischer Scientific). We loaded the protein from each compartment in proportion to the amount obtained (roughly 1:1:2 for chromatin:nucleoplasm:cytoplasm).
For mES cells, similar western-blot experiments were carried out using antibodies against actin (Abcam), histone H3 (Abcam) and SNRP70 (Abcam). We confirmed NAI-N3 treatment didn’t affect fractionation. Protein was quantified using the Pierce BCA protein assay kit (Thermo Fischer Scientific). We loaded the protein from each compartment in proportion to the amount obtained (roughly 1:1:2 for chromatin:nucleoplasm:cytoplasm).