Western blot analysis of extracts from 293T and Raw 264.7 cells, untreated (-) or treated with Nocodazole #2190 (100 ng/ml, 16 hr; +), using Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb (upper) and Histone H3 (D1H2) XP ® Rabbit mAb #4499 (lower).
Confocal immunofluorescent analysis of HeLa cells using Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye).
Flow cytometric analysis of Raw 264.7 cells using Phospho-Histone H3 (Thr4) (D5G1I) Rabbit mAb and Propidium Iodide (PI)/RNase Staining Solution #4087 to measure DNA content. Anti-rabbit IgG (H+L), F(ab') 2 Fragment (Alexa Fluor ® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 10 6 HeLa cells treated with Nocodazole #2190 (100 ng/ml) overnight, and either 5 μl of Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729, using SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP ® Human GAPDH Exon 1 Primers #5516, SimpleChIP ® Human α Satellite Repeat Primers #4486, and human D7Z1 satellite primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.