Journal: Cell chemical biology

Article Title: Hypomorph mutation-directed small-molecule protein-protein interaction inducers to restore mutant SMAD4-suppressed TGF-β signaling

doi: 10.1016/j.chembiol.2020.11.010

Figure Lengend Snippet: (A) Schematic illustration of the design of the TR-FRET assay for monitoring the SMAD4/SMAD3 PPI to discover small-molecule PPI inducers. Anti-Flag-Tb coupled with Flag-SMAD3 serves as the TR-FRET donor and anti-His-D2 coupled with His-SMAD4R361H serves as the acceptor. At the basal level, R361H impairs SMAD4 interaction with SMAD3, yielding low TR-FRET signal. Upon treatment with a PPI inducer, the induced SMAD3/SMAD4R361H complex formation brings two fluorophores into close proximity (<10 nm), generating a high TR-FRET signal.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tb cryptate-conjugated mouse monoclonal anti-Flag antibody Cisbio Bioassays Cat# 61FG2TLB D2-conjugated anti-HIS antibody Cisbio Bioassays Cat# 61HISDLF Mouse monoclonal anti-FLAG-HRP antibody Sigma-Aldrich Cat# A8592; RRID: AB_439702 Rabbit polyclonal anti-GST-HRP antibody Sigma-Aldrich Cat# A7340; RRID: AB_258340 Mouse monoclonal anti-β-Actin antibody Sigma-Aldrich Cat# A5441; RRID: AB_476744 Rabbit monoclonal anti-p21 antibody Cell signaling Cat# 2947S; RRID: AB_823586 Mouse monoclonal anti-p15 antibody Santa Cruz Cat# sc-271791; RRID: AB_10709436 SMAD4 (D3M6U) Rabbit mAb Cell signaling Cat# 38454; RRID: AB_2728776 SMAD3 mouse monoclonal Antibody (38-Q) Santa Cruz Cat# sc-101154; RRID: AB_1129525 Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) Cell signaling Cat# 93702; RRID: AB_2800208 Peroxidase AffiniPure Goat Anti-Rabbit IgG(H+L) secondary antibody Jackson ImmunoResearch Cat# 111–035-003 Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L) secondary antibody Jackson ImmunoResearch Cat# 115–035-003 Chemicals, Peptides, and Recombinant Proteins Recombinant Human TGF-beta 1 Protein R&D Systems Cat# 240-B Ro-31-8220 Cayman Cat# 138489–18-6 Go-6983 Cayman Cat# 133053–19-7 Ro-31-7549 Cayman Cat# 138516–31-1 Ro-31-8425 Cayman Cat# 145317–11-9 Ro-32-0432 Cayman Cat# 145333–02-4 UCN-1028c Cayman Cat# 121263–19-2 PKC-412 Cayman Cat# 120685–11-2 EEBL Emory ECBDC ( Mo et al., 2019 ) N/A FuGENE Promega Cat# E2311 SBE4-Luc gift from Bert Vogelstein ( Zawel et al., 1998 ) Addgene plasmid # 16495 Dual-Glo luciferase kit Promega Cat# E2920 Oligonucleotides p21-F: 5′-ATGTGTCCTGGTTCCCGTTTC-3′; IDT N/A p21-R: 5′-CATTGTGGGAGGAGCTGTGA-3′; IDT N/A p15-F: 5′- GGACTAGTGGAGAAGGTGCG-3′; IDT N/A p15-R: 5′-GGGCGCTGCCCATCATCATG-3′; IDT N/A GAPDH-F: 5′-GAAGGTGAAGGTCGGAGT-3′; IDT N/A GAPDH-R: 5′-GAAGATGGTGATGGGATTTC-3′.

Techniques:

Journal: Cell chemical biology

Article Title: Hypomorph mutation-directed small-molecule protein-protein interaction inducers to restore mutant SMAD4-suppressed TGF-β signaling

doi: 10.1016/j.chembiol.2020.11.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tb cryptate-conjugated mouse monoclonal anti-Flag antibody Cisbio Bioassays Cat# 61FG2TLB D2-conjugated anti-HIS antibody Cisbio Bioassays Cat# 61HISDLF Mouse monoclonal anti-FLAG-HRP antibody Sigma-Aldrich Cat# A8592; RRID: AB_439702 Rabbit polyclonal anti-GST-HRP antibody Sigma-Aldrich Cat# A7340; RRID: AB_258340 Mouse monoclonal anti-β-Actin antibody Sigma-Aldrich Cat# A5441; RRID: AB_476744 Rabbit monoclonal anti-p21 antibody Cell signaling Cat# 2947S; RRID: AB_823586 Mouse monoclonal anti-p15 antibody Santa Cruz Cat# sc-271791; RRID: AB_10709436 SMAD4 (D3M6U) Rabbit mAb Cell signaling Cat# 38454; RRID: AB_2728776 SMAD3 mouse monoclonal Antibody (38-Q) Santa Cruz Cat# sc-101154; RRID: AB_1129525 Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) Cell signaling Cat# 93702; RRID: AB_2800208 Peroxidase AffiniPure Goat Anti-Rabbit IgG(H+L) secondary antibody Jackson ImmunoResearch Cat# 111–035-003 Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L) secondary antibody Jackson ImmunoResearch Cat# 115–035-003 Chemicals, Peptides, and Recombinant Proteins Recombinant Human TGF-beta 1 Protein R&D Systems Cat# 240-B Ro-31-8220 Cayman Cat# 138489–18-6 Go-6983 Cayman Cat# 133053–19-7 Ro-31-7549 Cayman Cat# 138516–31-1 Ro-31-8425 Cayman Cat# 145317–11-9 Ro-32-0432 Cayman Cat# 145333–02-4 UCN-1028c Cayman Cat# 121263–19-2 PKC-412 Cayman Cat# 120685–11-2 EEBL Emory ECBDC ( Mo et al., 2019 ) N/A FuGENE Promega Cat# E2311 SBE4-Luc gift from Bert Vogelstein ( Zawel et al., 1998 ) Addgene plasmid # 16495 Dual-Glo luciferase kit Promega Cat# E2920 Oligonucleotides p21-F: 5′-ATGTGTCCTGGTTCCCGTTTC-3′; IDT N/A p21-R: 5′-CATTGTGGGAGGAGCTGTGA-3′; IDT N/A p15-F: 5′- GGACTAGTGGAGAAGGTGCG-3′; IDT N/A p15-R: 5′-GGGCGCTGCCCATCATCATG-3′; IDT N/A GAPDH-F: 5′-GAAGGTGAAGGTCGGAGT-3′; IDT N/A GAPDH-R: 5′-GAAGATGGTGATGGGATTTC-3′.

Techniques: Recombinant, Plasmid Preparation, Luciferase, Viability Assay, Software

Journal: Oncogene

Article Title: Cyclin D1 and D3 associate with the SCF complex and are coordinately elevated in breast cancer.

doi: 10.1038/sj.onc.1202511

Figure Lengend Snippet: Figure 1 Comparison of cyclin D1 and cyclin D3 protein and mRNA level in primary breast cancers samples. (a) Thirty mg of total protein were resolved on a 12% SDS ± PAGE. Anti-cyclin D1 rabbit polyclonal antibody (M20) was used for immunoblot- ting. The membrane was stripped and reprobed with anti-actin mouse monoclonal antibody (N350). RT ± PCR analysis in primary breast cancers of cyclin D1 (top panel) and actin (lower panel). Cyclin D1 RT ± PCR products were analysed by Southern blotting. (b) The same Western blotting membrane used for detecting cyclin D1 was stripped and reprobed using the anti- cyclin D3 polyclonal antibody C-16. RT ± PCR analysis of cyclin D3 (top panel) and actin (lower panel). Cyclin D3 RT ± PCR products were analysed by Southern blotting

Article Snippet: For detection of actin, the monoclonal antibody N350 (Amersham Life Science) was used at a dilution of 1 : 2000.

Techniques: Comparison, SDS Page, Western Blot, Membrane, Reverse Transcription Polymerase Chain Reaction, Southern Blot

Journal: Oncogene

Article Title: Cyclin D1 and D3 associate with the SCF complex and are coordinately elevated in breast cancer.

doi: 10.1038/sj.onc.1202511

Figure Lengend Snippet: Figure 4 (a) Eect of the proteosome inhibitor LLnL on cyclin D1 and D3 levels. U2-OS cells were treated with DMSO alone (7) or with LLnL in DMSO (+) for 14 h. Total proteins were extracted and 30 mg run on a 12% SDS ± PAGE. Immunoblotting was performed using anti-cyclin D1 rabbit polyclonal antibody (M20) and with anti-cyclin D3 polyclonal antibody (C-16). The membrane was stripped and reprobed with anti-actin mouse monoclonal antibody (N350). (b) Ubiquitination of cyclin D1 and D3. U2OS cells were transfected with pCDNA3-cyclin D1HA or pCDNA3-cyclin D3 plasmid alone or in combination with pCDNA3- ubiquitinMYC. Cyclin D1HA or cyclin D3 were immunoprecipitated and resolved on a 7.5% SDS ± polyacrylamide gel. The immunoblot was probed using the anti-Myc antibody 9E10 to detect ubiquitination of cyclin D1 and D3

Article Snippet: For detection of actin, the monoclonal antibody N350 (Amersham Life Science) was used at a dilution of 1 : 2000.

Techniques: SDS Page, Western Blot, Membrane, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Developmental Neuroscience

Article Title: Developmental Changes in the Expression of Lysophosphatidic Acid Receptor 4 in the Mouse Brain

doi: 10.1159/000550132

Figure Lengend Snippet: Expression of LPAR4 in the primary cultured mouse hippocampal neurons. The neurons were fixed at 3 div ( a ) or 7 div ( b ) and double-stained with anti-LPAR4 in together with anti-Tau ( a ) or anti-MAP2 ( b ). Merged images are also shown. Scale bars, 50 µm. Neurons were fixed at 14 div and double-stained with anti-LPAR4 together with anti-synaptophysin ( c ) or anti-PSD95 ( d ). The boxed area in each upper panel is magnified and shown in the lower panels. Scale bars, 20 µm (upper panels) and 2 µm (lower panels). e LPAR4 distribution in the dendrite. A representative image of a dendrite from a neuron fixed at 14 div is presented in the leftmost panel. Fluorescence profiles were measured along lines drawn in the image, and the results are shown in the three adjacent panels. Scale bar, 2 µm.

Article Snippet: The following mouse monoclonal antibodies were also used; anti-myc (Medical & Biological Laboratories), anti-Tau-1 (Millipore, Temecula, CA; USA), anti-MAP2 (Sigma-Aldrich, St. Louis, MO, USA), anti-GFAP (Sigma-Aldrich), anti-β-tubulin (Sigma-Aldrich), anti-synaptophysin (Progen Biotechnik, Heidelberg, Germany), and anti-PSD95 (Abcam, Tokyo, Japan).

Techniques: Expressing, Cell Culture, Staining, Fluorescence