Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: List of reagents and antibodies.

Article Snippet: 45 , Aurora B (AURKB) (NM_004217) Human Tagged ORF Clone , , RC210288 , OriGene.

Techniques: Concentration Assay, cDNA Synthesis, SYBR Green Assay, Protease Inhibitor, Saline, Plasmid Preparation, Magnetic Beads, Membrane

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: Bar graphs showing mitosis scores in the Emory ( A ) and Dekalb ( B ) cohorts. C Heatmap showing the expression levels of various kinases in the TCGA BC dataset. D , E Bar graphs showing the expression levels of PLK1 ( D ) and AURKB ( E ) in AA ( n = 3) and EA ( n = 3) TNBC cell lines. F – H Representative IHC images of PLK1 and AURKB ( F ) and quantification bar graphs showing PLK1 ( G ) and AURKB ( H ) levels in grade- and stage-matched AA and EA patients with TNBC (Dekalb cohort). I Immunoblot showing PLK1 and AURKB protein levels in AA and EA TNBC cell lines ( n = 3 each). FPKM fragments per kilobase of transcript per million mapped reads. Bars indicate mean ± SEM. Unpaired two-tailed Student’s t -test with Welch’s correction was used to determine statistical significance (* P < 0.05, *** P < 0.0005, ns = non-significant). The scale bar represents 100 µm.

Article Snippet: 45 , Aurora B (AURKB) (NM_004217) Human Tagged ORF Clone , , RC210288 , OriGene.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: Expression of various kinases in AA and EA patients with TNBC (TCGA dataset).

Article Snippet: 45 , Aurora B (AURKB) (NM_004217) Human Tagged ORF Clone , , RC210288 , OriGene.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: A , B Immunoblots showing the levels of PLK1, survivin, p-survivin (S20), and β-actin after PLK1 silencing ( A , B ) or inhibition ( C ) in AA and EA TNBC cell lines. D – F Immunoblots showing the levels of AURKB, survivin, p-survivin (T117), and β-actin after AURKB silencing ( D , E ), or inhibition ( F ) in AA and EA TNBC cell lines.

Article Snippet: 45 , Aurora B (AURKB) (NM_004217) Human Tagged ORF Clone , , RC210288 , OriGene.

Techniques: Western Blot, Inhibition

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: A – F Representative IHC images ( A , B ) and bar graphs ( C – F ) showing Ki-67 and survivin levels in AA ( C , E ) and EA ( D , F ) TNBC xenografts under various treatment conditions. G , H Immunoblots showing the levels of p-survivin (T117), p-survivin (S20), total survivin, AURKB, PLK1, and β-actin in AA ( G ) and EA ( H ) fresh-frozen xenograft tumor lysates from mice treated with volasertib, barasertib, or their combination ( n = 12 per treatment group). Bars represent mean ± SEM. Unpaired two-tailed Student’s t -test with Welch’s correction was used to determine statistical significance (**** P < 0.00005, ns = non-significant). The scale bar represents 100 µm.

Article Snippet: 45 , Aurora B (AURKB) (NM_004217) Human Tagged ORF Clone , , RC210288 , OriGene.

Techniques: Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson’s disease

doi: 10.1038/s41467-023-37464-2

Figure Lengend Snippet: A Schematic of the experimental design including proteomic screens, identification and validation of hits, target deconvolution, and evaluation in cell and animal models (created using Servier Medical Art at https://smart.servier.com/ ). For the cytotoxicity screen, we employed our peptide library to identify peptides that rescue cytotoxicity induced by a-syn overexpression and proteostatic stress (due to MG132 administration). To screen our library for peptides that inhibit a-syn oligomers, we used FACS with a split YFP-a-syn system (cells co-expressing V1S and SV2). B Validation of cell viability effect of peptides in A53T and WT a-syn expressing cells under proteostatic stress as well as controls. MG132 was used at a concentration of 10 μM. Experiments were done in triplicate. Data represent means ± s.d. (unpaired two-tailed t -tests; A53T PDpep1, P = 0.0051; A53T PDpep1.1, P = 0.0002; A53T PDpep1.2, P = 0.0035; A53T PDpep1.3, P < 0.0001; WT a-syn PDpep1.3, P = 0.0008; n = 3). C WT a-syn oligomers as measured by luciferase activity. All four peptides showed significant reduction in a-syn oligomers from cells stably expressing split luciferase-a-syn constructs. Experiments were done in triplicate. Data represent means ± s.d. (unpaired two-tailed t -tests; PDpep1, P = 0.0008; PDpep1.1, PDpep1.2, PDpep1.3, P < 0.0001; n = 3). D Co-immunoprecipitation experiment with Flag-CHMP2B and GFP-peptides ( n = 3). PDpep1 and PDpep1.3 peptides interacted with Flag-CHMP2B whereas GFP alone (CTL) did not. E Fluorescence polarization (FP) binding assay of a FITC-labeled PDpep1.3 peptide against CHMP2B. Error bars represent ± s.d. of the fit ( n = 5). F Knockdown of CHMP2B or VPS4 in A53T a-syn expressing cells under proteostatic stress. Cell viabilities were measured with cells stably expressing different shRNAs and transfected with Scramble or PDpep1.3 peptide. Experiments were done in triplicate. Data represent means ± s.d. (unpaired two-tailed t -tests; PDpep1.3, P = 0.0005; n = 3). ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Adeno-associated virus (AAV) of a 1/2 serotype was used to express A53T a-syn (AAV-A53T), a truncated version of A53T a-syn lacking amino acids 103–140 (Δa-syn(103–140)), and WT a-syn, fused to either the N-terminal half of YFP (AAV-V1S) or the C-terminal half of YFP (AAV-SV2), all under the control of the CAG promoter, a hybrid of the chicken beta-actin (CBA) promoter fused with the cytomegalovirus (CMV) immediate early enhancer sequence (GeneDetect Ltd.) .

Techniques: Over Expression, Expressing, Concentration Assay, Two Tailed Test, Luciferase, Activity Assay, Stable Transfection, Construct, Immunoprecipitation, Fluorescence, FP-binding Assay, Labeling, Transfection

Journal: Nature Communications

Article Title: Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson’s disease

doi: 10.1038/s41467-023-37464-2

Figure Lengend Snippet: A Representative immunoblots of a-syn levels in HEK293 cells expressing A53T a-syn plus GFP alone (CTL), peptides, or full-length CHMP2B (top panel). Loading control was beta-actin (middle panel). RT-PCR shows no change in a-syn mRNA levels (bottom panel). B Quantification of A53T a-syn protein level (normalized to beta-actin) from immunoblot data represented in ( A ). Bars represent means ± s.d. (unpaired two-tailed t -tests; PDpep1, PDpep1.2, PDpep1.3, CHMP2B, P < 0.0001; n = 3). C Quantification of a-syn mRNA from RT-PCR data represented in ( A ). Bars represent means ± s.d. ( n = 3). D Representative images of rat cortical neurons transduced with A53T a-syn plus Scramble1.3-GFP or PDpep1.3-GFP (scale bars = 5 µm). E Quantification of relative a-syn fluorescence in GFP-positive neurons. Bars represent means ± s.e.m. (two-tailed nested t -tests, t (55) = 6.545; P < 0.0001; n = 3). F Representative immunoblot of human a-syn (top panel) and GFP (middle panel) of lysates from cortical neurons transduced with A53T a-syn plus Scramble1.3-GFP or PDpep1.3-GFP. Loading control was beta-actin (bottom panel). G Quantification of relative endogenous a-syn fluorescence in GFP-positive neurons transduced with Scramble1.3-GFP or PDpep1.3-GFP. Bars represent means ± s.e.m. (two-tailed nested t -test, t (55) = 5.504; P < 0.0001; n = 3). H Representative images of rat cortical neurons transduced with V1S/SV2 or YFP plus Scramble1.3-RFP or PDpep1.3-RFP to assess for a-syn oligomer levels. Quantification of relative a-syn fluorescence in RFP-positive cortical neurons transduced with I V1S/SV2 plus Scramble1.3-RFP or PDpep1.3-RFP (two-tailed nested t -test, t (51) = 4.915; P < 0.0001; n = 3) or J V1S alone plus Scramble1.3-RFP or PDpep1.3-RFP (two-tailed nested t -test, t (36) = 3.468; P = 0.0014; n = 3). Quantification of relative YFP fluorescence in RFP-positive cortical neurons transduced with K V1S/SV2 plus Scramble1.3-RFP or PDpep1.3-RFP (two-tailed nested t -test, t (54) = 5.455; P = 0.0055; n = 3) or L YFP plus Scramble1.3-RFP or PDpep1.3-RFP (two-tailed nested t -test, t (56) = 0.092; P = 0.93; n = 3). M Representative images of cortical neurons treated with human a-syn PFFs plus Scramble1.3-GFP or PDpep1.3-GFP (scale bars = 5 μm). N Quantification of number of pS129 a-syn + puncta per GFP-positive neuron (unpaired two-tailed t -test, t (4) = 3.099; P = 0.0362; n = 3). * P < 0.05; ** P < 0.01; **** P < 0.0001; ns indicates P > 0.05. Source data are provided as a Source Data file.

Article Snippet: Adeno-associated virus (AAV) of a 1/2 serotype was used to express A53T a-syn (AAV-A53T), a truncated version of A53T a-syn lacking amino acids 103–140 (Δa-syn(103–140)), and WT a-syn, fused to either the N-terminal half of YFP (AAV-V1S) or the C-terminal half of YFP (AAV-SV2), all under the control of the CAG promoter, a hybrid of the chicken beta-actin (CBA) promoter fused with the cytomegalovirus (CMV) immediate early enhancer sequence (GeneDetect Ltd.) .

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Transduction, Fluorescence

Journal: Nature Communications

Article Title: Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson’s disease

doi: 10.1038/s41467-023-37464-2

Figure Lengend Snippet: A Representative image of an adult C. elegans with PDE neuron visualized using dat-1p::gfp ( egIs1 ) (scale bar = 20 μm) (top panel). Neurite length for each PDE neuron was categorized as: Short (does not extend past the vulva; purple), Medium (extends past the vulva but not beyond halfway to ADE neuron; cyan), or Long (extends to ADE neuron; yellow) (created using an image from somersault1824 at https://somersault1824.gumroad.com/ ). B Frequencies of PDE neurite lengths were compared for C. elegans expressing no a-syn versus a-syn alone (Pearson Chi-Square test, χ 2 (2, 1252) = 169.0; P < 0.0001), a-syn with RFP (a-syn;TagRFP Marker+) versus a-syn without RFP (a-syn;TagRFP Marker-) (Pearson Chi-Square test, χ 2 (2, 910) = 1.332; P = 0.5137), and a-syn with RFP-PDpep1.3 (a-syn;TagRFP::PDpep1.3 Marker+) versus a-syn without RFP-PDpep1.3 (a-syn;TagRFP::PDpep1.3 Marker-) (Pearson Chi-Square test, χ 2 (2, 1528) = 86.1729; P < 0.0001). C Representative images of native YFP and RFP fluorescence and immunostaining with anti-tyrosine hydroxylase (TH) antibody in substantia nigra (SN) of rats injected with V1S/SV2 or YFP plus Scramble1.3-RFP or PDpep1.3-RFP (scale bars = 200 μm). Quantification of YFP + area in SN of rats injected with D V1S/SV2 (unpaired two-tailed t -test, t (16) = 2.317; P = 0.0341; Scramble1.3, n = 8 rats; PDpep1.3, n = 10 rats) or E full-length YFP (unpaired two-tailed t -test, t (15) = 1.003; P = 0.3317; Scramble1.3, n = 8 rats; PDpep1.3, n = 9 rats). Quantification of YFP + area in striatum of rats injected with F V1S/SV2 (unpaired two-tailed t -test, t (16) = 2.193; P = 0.0434; Scramble1.3, n = 8 rats; PDpep1.3, n = 10 rats) or G full-length YFP (unpaired two-tailed t -test, t (13) = 1.140; P = 0.2749; Scramble1.3, n = 6 rats; PDpep1.3, n = 9 rats). H Quantification of a-syn + area in SN (unpaired two-tailed t -test, t (16) = 2.843; P = 0.0118; Scramble1.3, n = 8 rats; PDpep1.3, n = 10 rats), I TH + cell counts in SN (unpaired two-tailed t -test, t (15) = 2.421; P = 0.0286; Scramble1.3, n = 8 rats; PDpep1.3, n = 9 rats), and J TH fluorescence in striatum (unpaired two-tailed t -test, t (16) = 2.664; P = 0.0170; Scramble1.3, n = 8 rats; PDpep1.3, n = 10 rats) at 6 weeks post-injection of V1S/SV2 plus Scramble1.3-RFP or PDpep1.3-RFP. Bars represent means ± s.e.m. * P < 0.05; **** P < 0.0001; ns indicates P > 0.05. Source data are provided as a Source Data file.

Article Snippet: Adeno-associated virus (AAV) of a 1/2 serotype was used to express A53T a-syn (AAV-A53T), a truncated version of A53T a-syn lacking amino acids 103–140 (Δa-syn(103–140)), and WT a-syn, fused to either the N-terminal half of YFP (AAV-V1S) or the C-terminal half of YFP (AAV-SV2), all under the control of the CAG promoter, a hybrid of the chicken beta-actin (CBA) promoter fused with the cytomegalovirus (CMV) immediate early enhancer sequence (GeneDetect Ltd.) .

Techniques: Expressing, Marker, Fluorescence, Immunostaining, Injection, Two Tailed Test

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Nicotinamide Adenine Dinucleotide-Dependent Binding of the Neuronal Ca 2+ Sensor Protein GCAP2 to Photoreceptor Synaptic Ribbons

doi: 10.1523/JNEUROSCI.3701-09.2010

Figure Lengend Snippet: Antibodies and labeling reagents

Article Snippet: More details on the antibodies used are given in . table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies/reagents Source Dilution used Secondary antibody Dilution used Antibodies used for Western blotting GCAP2 6th immune serum Rabbit polyclonal 1:1000 GAR-POX; Sigma, cat. #A6154 1:10,000 GCAP2(A1); Santa Cruz Biotechnology, cat. #SC-59543 Mouse monoclonal 1:1000 GAM-POX; Sigma, cat. #A3673 1:10,000 U2656 ( Schmitz et al., 2000 ) Rabbit polyclonal 1:10,000 GAR-POX; Sigma, cat. #A6154 1:10,000 RIBEYE/CtBP2; BD Transduction Laboratories, cat. #612044 Mouse monoclonal 1:10,000 GAM-POX; Sigma, cat. #A3673 1:10,000 Antibodies used for immunolabeling GCAP2 6th immune serum Rabbit polyclonal 1:500 GAR-Cy3; Zymed, cat. #81-6115 1:1000 GCAP2(A1); Santa Cruz Biotechnology, cat. #SC-59543 Mouse monoclonal 1:1000 GAM-Cy3; Rockland, cat. #610-104-121 1:1000 U2656 ( Schmitz et al., 2000 ) Rabbit polyclonal 1:1000 GAR-Cy2; Rockland, cat. #611-111-122 1:1000 RIBEYE/CtBP2; BD Transduction Laboratories, cat. #612044 Mouse monoclonal 1:500 GAM-Cy2; Jackson ImmunoResearch, cat. #115-096-146 1:1000 mGluR6; Neuromics/Acris, cat. #RA13105 Rabbit polyclonal 1:500 GAR-Cy3; Sigma, cat. #C 2821 1:1000 SV2 A; Synaptic Systems, cat. #119 00 2 Rabbit polyclonal 1:500 GAR-Cy2; Rockland, cat. #611-111-122 1:1000 Synaptophysin; Sigma, cat. #S5768 Mouse monoclonal 1:500 GAM-Cy2; Jackson ImmunoResearch, cat. #115-096-146 1:1000 Antibodies used for whole-mount immunostaining U2656 ( Schmitz et al., 2000 ) Rabbit polyclonal 1:500 GAR-Cy3; Zymed, cat. #81-6115 1:1000 Reagent used for PNA labeling Lectin PNA from Arachis hypogaea a Lectin PNA; Invitrogen cat. #L-21409 1:250 Open in a separate window Antibodies used for in situ proximity ligation assays are summarized in detail in Materials and Methods. cat., Catalog; GAR, goat anti-rabbit; GAM, goat anti-mouse; PNA, peanut agglutinin. a Alexa Fluor 488 conjugate.

Techniques: Labeling, Western Blot, Transduction, Immunolabeling, Immunostaining