Journal: bioRxiv

Article Title: CCDC32 stabilizes clathrin-coated pits and drives their invagination

doi: 10.1101/2024.06.26.600785

Figure Lengend Snippet: (A) AP2 structure. AD: appendage domain. (B-C) Immunoprecipitation (IP) of eGFP from naive ARPE-HPV cells (control IPs) or ARPE-HPV cells that stably express AP2-α-eGFP (AP2-α-eGFP IPs) using anti-GFP beads. (B) The domain structure of AP2-α-eGFP. (C) Volcano plot after mass spectrometry analysis of the IP samples for protein ID and abundance detection. Green: AP2 subunits; orange: known AP2 interactors. (D-F) GST pull-down assays. (D) Coomassie blue stained SDS-page gel of purified GST, GST-AP2-α-AD, and GST-AP2-β-AD. (E) Representative western blot result of n=3 GST pull-down assay from ARPE-HPV eGFP-CCDC32(FL) cell lysate using purified GST, GST-AP2-α-AD, or GST-AP2-β-AD. The amount of bait GST-proteins is shown in (D), and the pulled down eGFP-CCDC32 was detected by immunoblotting (IB) of GFP. (F) Relative CCDC32 enrichments quantified from immunoblotting results as shown in (E). (G-I) IP of eGFP from ARPE-HPV cells that stably express eGFP or eGFP-CCDC32(FL) using anti-GFP magnetic beads. (G) The domain structures of eGFP and eGFP-CCDC32(FL). (H) Representative immunoblotting result of n=3 IP samples. (I) Relative AP2 enrichments quantified from immunoblotting results. Error bars are SD. Two-tailed student’s t-test: ***, p ≤ 0.001.

Article Snippet: GST in a pGEX-6P-1 vector, GST-AP2-α-AD in a pGEX-2T-1 vector, and GST-AP2-β-AD in a pGEX-4T-1 vector were transfected and expressed in BL21(DE3) separately, and then affinity purified using GSTrap HP column (Cytiva).

Techniques: Immunoprecipitation, Control, Stable Transfection, Mass Spectrometry, Staining, SDS Page, Purification, Western Blot, Pull Down Assay, Magnetic Beads, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 1. BRD4 NCC loss of function produces severe craniofacial phenotypes.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 2. BRD4 mutant mandibular cNCCs fail to properly differentiate to osteoblast lineages.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Mutagenesis

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 3. Loss of BRD4 disrupts in vitro cNCCs osteoblast differentiation.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: In Vitro

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 4. BRD4 binds to proximal active enhancers to regulate osteogenic transcription.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 5. BRD4 directly regulates transcription of factors critical for osteoblast differentiation.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 6. BRD4 associates with RUNX2 to regulate osteoblast differentiation

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 7. Model of BRD4 function in CdLS craniofacial pathogenesis (created with

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Spt5 Phosphorylation and the Rtf1 Plus3 Domain Promote Rtf1 Function through Distinct Mechanisms

doi: 10.1128/MCB.00150-20

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: Plus3 domain point mutations were introduced using the Phusion site-directed mutagenesis kit (Thermo Fisher Scientific) and verified by sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Plasmid Description Reference or source pJT9 pFA6a-kanMX6-CTAP2 48 pJT48 pGEX-6P-1 Addgene pJT118 pET StrepII tag LIC cloning vector (2RT) Addgene pJT123 pET His6 TEV tag LIC cloning vector (1B) Addgene pJT124 pGEX-6P-1-Prf1-His6 (expresses GST-Prf1-His, inserted at BamHI/EcoRI) This study pJT125 pGEX-6P-1-N-Terminal domain of Prf1 (expresses GST–N-terminal domain of Prf1, inserted at BamHI/XhoI) This study pJT126 pGEX-6P-1-plus3 domain of Prf1 (expresses GST-Plus3 domain of Prf1, inserted at BamHI/XhoI) This study pJT127 pGEX-6P-1-C-Terminal domain of Prf1 (expresses GST–C-terminal domain of Prf1, inserted at BamHI/XhoI) This study pJT129 pGEX-6P-1-(+3+C) domains of Prf1 (expresses GST-+3+C fragment of Prf1, inserted at BamHI/XhoI) This study pJT130 pGEX-6P-1-(+3 domainR227A of Prf1) (expresses GST-Plus3 domain R227A point mutant, inserted at BamHI/XhoI) This study pJT131 pGEX-6P-1-(+3 domainR262E of Prf1) (express GST-Plus3 domain R262E point mutant, inserted at BamHI/XhoI) This study pJT132 pGEX-6P-1-(+3 domainR296E of Prf1) (expresses GST-Plus3 domain R296E point mutant, inserted at BamHI/XhoI) This study pJT150 pGex-6p-1-Rtf1-TAP-kanMX6 (inserted at XhoI/EagI) This study pJT152 pET His6 TEV LIC-Cdc73 (expresses His-Cdc73) This study pJT153 pET His6 TEV LIC-Leo1 (expresses His-Leo1) This study pJT154 pET His6 TEV LIC-Tpr1N (1-400aminoacids) (expresses His-Tpr1N) This study pJT161 pGex-6p-1-Rtf1-R227A-TAP-kanMX6 (inserted at XhoI/EagI) This study pJT162 pGex-6p-1-Rtf1-R262E-TAP-kanMX6 (inserted at XhoI/EagI) This study pJT163 pGex-6p-1-Rtf1-R296E-TAP-kanMX6 (inserted at XhoI/EagI) This study pJT175 pET Strep-tag vector (p118) C-terminus of Paf1 (aa 260–456) (expresses Strep-tag II–Paf1C) This study Open in a separate window Plasmids used in this study To integrate mutations into the chromosomal prf1 + locus, an ∼4.5-kb region spanning the locus and including ∼250 bp of 5′ and 3′ homology was PCR amplified from strain JT202 ( prf1-TAP :: kanMX6 ) and cloned into pGEX-6P-1 to create pJT150.

Techniques: Cloning, Plasmid Preparation, Mutagenesis, Strep-tag