Protein Extraction and Western Blot Analysis

Total proteins were extracted from the top sixth leaves [51 (link)], the cytoplasmic fraction, and the nuclear fraction [52 (link)] of 50-day-old plants grown in the chamber. Western blots were hybridized with the specific antibody against YFP (Novagen) or the specific NtTTG2 antibody, which was produced by immunizing a New Zealand white rabbit in our previous study [47 (link)]. PEPC and histone H3 that were used as cytoplasmic and nuclear markers [4 (link), 41 (link)] were hybridized with a specific PEPC antibody (Rockland) and histone H3 antibody (Abcam), respectively. Hybridized blots were probed by using a horseradish peroxidase-conjugated secondary antibody (Beyotime) according to the manufacturer’s recommendations.

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Ge J., Li B., Shen D., Xie J., Long J, & Dong H. (2016). Tobacco TTG2 regulates vegetative growth and seed production via the predominant role of ARF8 in cooperation with ARF17 and ARF19. BMC Plant Biology, 16, 126.

Publication 2016

histone H3 antibody horseradish peroxidase-conjugated secondary antibody

Antibody Cytoplasmic Histone h3 Horseradish peroxidase New zealand white rabbit Plants Proteins Western blots

Corresponding Organization :

Other organizations : Nanjing Agricultural University

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Variable analysis

independent variables

Fraction of proteins extracted (total proteins, cytoplasmic fraction, nuclear fraction)

dependent variables

Presence and levels of YFP and NtTTG2 proteins as detected by Western blot

control variables

Age of plants (50-day-old)
Growth conditions (chamber)
Antibodies used (anti-YFP, anti-NtTTG2, anti-PEPC, anti-histone H3)
Experimental techniques (Western blot, hybridization, secondary antibody)

controls

Positive controls: PEPC and histone H3 as cytoplasmic and nuclear markers, respectively
Negative control: Not explicitly mentioned

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