Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with constructs expressing Myc/DDK-tagged full-length human cyclin D3 (hCyclin D3-Myc/DDK) or a threonine to alanine point mutation at amino acid 283 (hCyclin D3-T283A-Myc/DDK), using Phospho-Cyclin D3 (Thr283) (E1V6W) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Western blot analysis of extacts from ACHN cells, untreated or treated with UV light (100 mJ/cm2) with recovery times as indicated, using Phospho-Cyclin D3 (E1V6W) Rabbit mAb (upper), total Cyclin D3 (DCS22) Mouse mAb #2936 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from Raji or Gumbus cell lines, untreated (-) or treated with MG-132 #2194 (10 μM, overnight; +), using Phospho-Cyclin D3 (Thr283) (E1V6W) Rabbit mAb or β-Actin (D6A8) Rabbit mAb #8457 (lower). Gumbus cells have a genetic mutation in cyclin D3 at Thr283.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
Phospho-Cyclin D3 (Thr283) (E1V6W) Rabbit mAb recognizes endogenous levels of cyclin D3 protein only when phosphorylated at Thr283. Bands of unknown origin are detected at 140 kDa and higher.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Thr283 of human cyclin D3 protein.
Activity of the cyclin-dependent kinases CDK4 and CDK6 is regulated by T-loop phosphorylation, by the abundance of their cyclin partners (the D-type cyclins), and by association with CDK inhibitors of the Cip/Kip or INK family of proteins (1). The inactive ternary complex of cyclin D/CDK4 and p27 Kip1 requires extracellular mitogenic stimuli for the release and degradation of p27 concomitant with a rise in cyclin D levels to affect progression through the restriction point and Rb-dependent entry into S-phase (2). The active complex of cyclin D/CDK4 targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (3). Levels of cyclin D protein drop upon withdrawal of growth factors through downregulation of protein expression and phosphorylation-dependent degradation (4).
Although the D-type cyclins are not fully redundant, cyclin D3, like D1, plays a prominent role in differentiation and proliferation, which correlates with higher expression levels of cyclin D3 in various cancers (5). Burkitt lymphoma can carry mutations in the cyclin D3 gene that can affect phosphorylation at Thr283, stability of the protein, and cell cycle progression (6).
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