Western blot analysis of extracts from various cell lines using Phospho-SF3B1 (Thr313) (D8D8V) Rabbit mAb.
Western blot analysis of extracts from HeLa cells, untreated (-) or treated with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) (+) using Phospho-SF3B1 (Thr313) (D8D8V) Rabbit mAb (upper) or SF3B1 (D7L5T) Rabbit mAb #14434 (lower).
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-SF3B1 (Thr313) (D8D8V) Rabbit mAb recognizes endogenous levels of SF3B1 protein only when phosphorylated at Thr313.Species Reactivity:
Human, MouseSpecies predicted to react based on 100% sequence homology:
Hamster, Xenopus, Zebrafish
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr313 of human SF3B1 protein.
Splicing factor 3b subunit 1 (SF3B1) is an integral component of the U2 small nuclear ribonucleoprotein (U2 snRNP) and plays an important role in the splicing of pre-mRNA that involves the removal of introns and the joining of exons to form mature mRNA (1-3). The assembly and proper recognition of splice sites are driven by sequences at the pre-mRNA intron-exon splice sites. The 5’ splice donor site is recognized by the U1 snRNP complex, while U2 snRNP binds to the 3’ splice site (branch point), ensuring the anchoring of the spliceosome machinery at the splice sites (3,4). Recent whole exome sequencing studies have demonstrated a high incidence of somatic mutations of SF3B1 in patients with various hematological malignancies such as chronic lymphocytic leukemia and myelodysplastic syndromes (2,3,5,6). Misregulation of pre-mRNA splicing arising from mutations of the spliceosome components such as SF3B1 is thought to contribute to changes in the expression patterns of key proteins that are involved in pathways such as cell cycle progression, cell death, and cancer metabolism (2,3).
Phosphorylation of SF3B1 at Thr313 is only found in catalytically active spliceosomes and associates mainly with chromatin, where about 80% of pre-mRNA splicing occurs. Treatment with a transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) leads to a decreased supply of pre-mRNA, resulting in the loss of phospho-SF3B1 (Thr313), consistent with its association with active splicing (7).
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