Journal: The Journal of Biological Chemistry

Article Title: Cancer-relevant Splicing Factor CAPERα Engages the Essential Splicing Factor SF3b155 in a Specific Ternary Complex *

doi: 10.1074/jbc.M114.558825

Figure Lengend Snippet: Domains of UHM and ULM-containing proteins relevant to this study. A, human CAPERα (NCBI RefSeq NP_004893) compared with human paralogues CAPERβ (NP_060577), U2AF65 (NP_009210), Puf60 (NP_001258027), and SPF45 (NP_001139019). B, human ULM-containing splicing factors SF1 (NP_004621) and SF3b155 (NP_036565). Circled P, phosphorylated SF1 SPSP motif. HEAT, helical repeats; KH-QUA2, K-Homology Quaking-Homology-2; RS, arginine-serine-rich; RRM, RNA recognition motif (blue); UHM, U2AF homology motif (cyan); ULM, U2AF ligand motif (magenta); Zn, zinc knuckle. Sequence boundaries of domains relevant to this study and the residue numbers of C termini are indicated above. C, ULM “consensus” compared with known sequences of human splicing factor ULMs. ULM tryptophans are highlighted in yellow.

Article Snippet: For experiments with cell extracts, bound proteins were analyzed by SDS-PAGE and immunoblotting with anti-U2AF 65 (mouse mAb, clone MC3; Sigma), anti-CAPERα (mouse mAb P14; Santa Cruz), and anti-GST (mouse mAb B14; Santa Cruz Biotechnology).

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Cancer-relevant Splicing Factor CAPERα Engages the Essential Splicing Factor SF3b155 in a Specific Ternary Complex *

doi: 10.1074/jbc.M114.558825

Figure Lengend Snippet: Isothermal titration calorimetry of CAPERα UHM binding ULMs or ULM-containing proteins Average values and S.D. of two independent experiments. Δ G ° was calculated using Δ G ° = −RTln( K D −1 ), and − T Δ S ° was calculated using Δ G ° = Δ H ° − T Δ S °, T = 303 K. Values for each class of multiple sites in the wild-type SF3b155 domain describe binding of one CAPERα UHM to one of these SF3b155 sites. Representative isotherms are given with c-values in supplemental Fig. 1 . Boundaries of SF3b155, -W293, -W338 are residues 190–344; ULM5 is residues 333–342 (KRKSRWDETP); ULM5L is residues 333–355 (KRKSRWDETPASQMGGSTPVLTP).

Article Snippet: For experiments with cell extracts, bound proteins were analyzed by SDS-PAGE and immunoblotting with anti-U2AF 65 (mouse mAb, clone MC3; Sigma), anti-CAPERα (mouse mAb P14; Santa Cruz), and anti-GST (mouse mAb B14; Santa Cruz Biotechnology).

Techniques: Isothermal Titration Calorimetry, Binding Assay

Journal:

Article Title: In Vivo Requirement of the Small Subunit of U2AF for Recognition of a Weak 3? Splice Site ▿ †

doi: 10.1128/MCB.00350-06

Figure Lengend Snippet: RNAi-mediated knockdown of U2AF subunits. Western blot analysis of HeLa cell lysates prepared 48 h after transfection with siRNAs against luciferase (GL2) (lane 1), U2AF65 (lane 2), U2AF35a (lane 3), U2AF35b (lane 4), and U2AF35a simultaneously with U2AF35b (lane 5). The blot was probed with antibodies against U2AF65, U2AF35, and β-actin, as indicated. Molecular mass markers are shown on the left.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibodies directed against U2AF 65 (MC3) ( 8 ), β-actin (clone AC-15; Sigma), and the FLAG epitope (M2; Sigma) and rabbit polyclonal sera directed against U2AF 35 (kindly provided by Angus Lamond [ 3 ]) and the HA epitope (Y-11; Santa Cruz Biotechnology).

Techniques: Western Blot, Transfection, Luciferase

Journal: RNA

Article Title: The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells

doi: 10.1261/rna.037069.112

Figure Lengend Snippet: Efficient CBC knockdown does not affect snRNP levels. HeLa cells transduced with retroviral vectors expressing CBP80 shRNA or without shRNA (Control) were assayed on day 6. ( A ) Assessment of CBC depletion through semiquantitative Western blotting ( left panel) of CBP80, CBP20, and GAPDH after CBP80 knockdown (KD). Decreasing amounts of the same lysate were loaded. Comparison of changes in CBP80 and CBP20 RNA and protein levels ( right panel). Band intensities were measured as integrated densities from Western blots and normalized to GAPDH. RNA levels were determined by RT-qPCR and normalized to 18S rRNA. N ≥ 8 different knockdowns. Error bars are the SEM. The changes are statistically significant ( P < 0.005), as determined by the Student’s t -test. ( B ) Immunoprecipitation of snRNPs with α-TMG (K121), α-Sm proteins (Y12), and α-SART3. RNA was extracted, resolved on a 10% urea gel, and detected by Northern blot. A longer exposure of α-TMG IP lanes is shown.

Article Snippet: The following mouse monoclonal antibodies were used: α-GAPDH (Novus Biologicals); α-RNA Pol II CTD, 4H8 (Abcam); α-TMG, K121 (Calbiochem); α-U2AF-65, MC3 ( ); and α-Sm proteins, Y12 ( ).

Techniques: Transduction, Expressing, shRNA, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Northern Blot

Journal: PLoS ONE

Article Title: The Protein Kinase KIS Impacts Gene Expression during Development and Fear Conditioning in Adult Mice

doi: 10.1371/journal.pone.0043946

Figure Lengend Snippet: A. Expression in different neonate tissues. 10 µg of total proteins were loaded on 10% acrylamide SDS PAGE and analyzed by Western blot using different antibodies. B. Western blots quantification. Expression of KIS and SF1 were ubiquitous but higher in brain. As expected β-tubulin levels were higher in brain and β-actin was lower in skeletal muscle and heart . C. Expression in brain during development. The profile of expression of proteins during brain development was assessed by western blot. Brains extracts from neonate and adult brains of KIS-ko animals were used to control the specificity of the anti KIS antibodies (right panel). D. Western blot quantification. The signal for the different proteins was normalized to β-actin and to the mean of E11 to E13 signals. Similar profiles were observed for splicing factors SF1 and U2AF 65 which are known to functionally interact and for the U2snRNP component SAP155 that interacts with U2AF 65 and KIS , .

Article Snippet: Commercial antibodies were mouse anti-U2AF 65 (clone MC3; Sigma), mouse anti-β-actin (clone AC-15; Sigma), mouse anti-β-tubulin (clone E7, Developmental Studies Hybridoma Bank) and mouse anti-SAP155 (clone D221-3; MBL).

Techniques: Expressing, SDS Page, Western Blot