Figure 4.
Figure 4. Transcription is required for full incorporation of new dCENP-A. (A) Experimental setup for chromatin fractionations on high- and low-salt sucrose step gradients analyzed in C and D. Fractions 1–3 contain soluble proteins; fractions 8–10 represent the chromatin fractions. o/n, overnight. (B) Chromatin of differentially treated cells stably expressing TI-dCENP-AHA fractionated on a 10/50% sucrose step gradient. Chromatin was monitored by absorbance at 254 nm. (C and D) Western blot after separation on low-salt (C) or high-salt (D) step gradients. Treatment of sample is indicated on the left, and each sample was probed for TI-dCENP-AHA (upper/α HA) and H1 (lower/α H1). Behavior of histone H3 is depicted for control treatment at the bottom. Arrows mark protein of interest and asterisks mark unspecific bands or potential degradation products.

Transcription is required for full incorporation of new dCENP-A. (A) Experimental setup for chromatin fractionations on high- and low-salt sucrose step gradients analyzed in C and D. Fractions 1–3 contain soluble proteins; fractions 8–10 represent the chromatin fractions. o/n, overnight. (B) Chromatin of differentially treated cells stably expressing TI-dCENP-AHA fractionated on a 10/50% sucrose step gradient. Chromatin was monitored by absorbance at 254 nm. (C and D) Western blot after separation on low-salt (C) or high-salt (D) step gradients. Treatment of sample is indicated on the left, and each sample was probed for TI-dCENP-AHA (upper/α HA) and H1 (lower/α H1). Behavior of histone H3 is depicted for control treatment at the bottom. Arrows mark protein of interest and asterisks mark unspecific bands or potential degradation products.

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