Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: An Ultrapotent, Ultraeconomical, Antifreeze Polypeptide

doi: 10.1002/adma.202420504

Figure Lengend Snippet: Polypeptide structural characterization. A) FTIR traces of t Bu‐E L NCA as compared to (A L E L ) 50 . B) FTIR traces of protecting‐group‐free E L NCA as compared to (A L E L ) 50 . C–E) CD spectra of polypeptides in PBS buffer at 20 °C. Data in C) indicates length dependent helical propensity of (A L E L ) n ; D) reveals that (A L E L ) 50 and (A L K L ) 50 adopt right‐handed α‐helices similar to that of winter flounder AFP1 but (V L E L ) 50 is disordered; and E) indicates that mirror‐image (A D E D ) 50 adopts a left‐handed α‐helix while racemic (A L/D E L/D ) 50 is disordered. F) CD spectra in PBS buffer (average of three scans) of (A L E L ) 50 at 4, 95, and at 4 °C after heating, demonstrating that the structure is thermally reversible and, by comparison to 20 °C data in E, that helical order increases slightly as the solution approaches freezing.

Article Snippet: For the primary antibody, α‐EGFP (rabbit polyclonal) in PBS (Cat. No.: 16118‐T16) was purchased from SinoBiological.

Techniques: Circular Dichroism, Comparison

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: GFP and mCherry protein accumulation in HEK293 cells transduced with PTRE-RiboTag lentivirus were confirmed by (A) microscopy and (B) Western blot. Lysate – total soluble protein. Sup – supernatant. IP – immunoprecipitated protein

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Transduction, Microscopy, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: (A) Total soluble protein from co-culture lysates, supernatants (Sup), and immunoprecipitated (IP) protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to HA or V5. (B) mCherry and GFP mRNA concentration was measured in triplicate by reverse transcription digital PCR in RNA isolated from the co-culture lysates (yellow), HA IP (blue), and V5 IP (purple), and plotted as a ratio. (C) RPL22-V5 and RPL22-HA were simultaneously IPed from co-culture lysates and selectively eluted in the indicated order using V5 or HA peptides, respectively. Total soluble protein from co-culture lysate and IPed protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to V5 or HA. (D) RPL22-V5 and RPL22-HA were sequentially IPed in the indicated order from co-culture lysates. Total soluble protein from co-culture lysates and IPed protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to HA or V5.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Co-Culture Assay, Immunoprecipitation, SDS Page, Concentration Assay, Digital PCR, Isolation

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: (A) mCherry and GFP mRNA concentration was measured in triplicate by reverse transcription digital PCR in RNA isolated from co-culture lysates (yellow), HA IP (blue), and V5 IP (purple), and plotted as a ratio. (B) RNA-seq reads were mapped to a hybrid reference genome containing hg38 and mm20 chromosomes and quantified by species for monocultures, co-culture, and IP samples. (C) Spearman correlation plot of RNA-seq for co-culture and IP samples. (D) The change in uniquely mapping reads (IP – input) for each chromosome was plotted against the initial (Input) percent uniquely mapping reads. Each dot represents a chromosome (hg38 – blue, mm20 – purple). First and second IPs are represented by open and filled dots, respectively. Chromosomes that fall above the red dotted line are enriched whereas chromosomes that fall below the red dotted line were depleted in IP samples compared to the Input. (E) The change in on-target (e.g. hg38 for HA IP and mm20 for V5) percent uniquely mapping reads (IP – Input) was plotted against the initial (Input) percent uniquely mapping reads at the gene level. Protein coding genes are in cyan and non-protein coding genes in pink. First and second IPs are represented by circles and triangles, respectively. Mitochondrially encoded genes have additional black colored fill.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Concentration Assay, Digital PCR, Isolation, Co-Culture Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: The ratio of mCherry to GFP transcript abundance was measured in triplicate from pre- and post-IP samples and reported as a ratio of the average concentration. IPs were performed in both orders as indicated.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: (A-B) RPKM of reporter gene expression (GFP/mCherry) in primary mouse astrocytes and hiPSC-derived motor neurons split by (A) RiboTag and (B) cell type. RPKM was calculated using reads mapped to GFP or mCherry and a total library size of reads mapping to the on-target cell type. (C) RNA-seq reads were mapped to a hybrid reference genome containing hg38 and mm20 chromosomes and quantified by species for co-cultures and IP samples. Matched samples are indicated by replicate number. (D) mCherry and GFP levels were measured by RNA-seq. The ratio of mCherry to GFP is reported for co-cultures and IP samples from hiPSC-MNs (MN IP) and primary mouse astrocytes (mAstro IP). HA RiboTag (purple). V5 RiboTag (blue). Lines indicate matched IPs and co-cultures.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Expressing, Derivative Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: (A) Scatter plot of on-target reporter (RPL22-HA-GFP; RPL22-V5-mCherry) gene expression in RPKM versus depletion efficiency. RPKM was calculated using the library size for the indicated cell type (i.e. only human reads were used to calculate RPKM for reporter expression in hiPSC-MNs and vice versa). (B) Scatter plot of the initial species composition of the coculture (left – human; right – mouse) versus the corresponding depletion efficiency for every IP. The on-target cell type is indicated by color (mAstro – purple; hiPSC-MN – blue) and shape indicates the RiboTag (HA – circles, V5 – triangles). For example, blue circles & triangles on the left examine the relationship between the depletion efficiency and the relative composition of the on-target cell type (human) in the initial coculture. The purple circles on the left examine the relationship between the depletion efficiency and the off-target cell type (mouse). The reverse is true for the plot on the right.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Expressing

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: Plotted is the ratio of mCherry to GFP across co-cultures (yellow), V5 IPs (blue), and HA IPs (purple) for (A) NGN2-induced excitatory neuron (iGluN) and ASCL1/DLX2-induced GABAergic neuron (iGaNs) co-cultures prepared from a single differentiation and RiboTag transduction per cell type, and (B) primary mouse astrocytes (mAstro) and hiPSC-MN co-cultures across independent replicates. Lines indicated matched IPs and co-cultures.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Transduction

Journal: Virology Journal

Article Title: Molecular characterization of duck enteritis virus UL41 protein

doi: 10.1186/s12985-018-0928-4

Figure Lengend Snippet: DEV-UL47 protein may translocate pUL41 into the nucleus in the absence of other viral proteins. All transfected samples were collected after 24 h post transfection. a DEF cells were transfected with pEGFP-C2-UL41 and pEGFP-C2, respectively. The EGFP-UL41 fusion protein was localized to the cytoplasm (green), further detected with mouse anti-UL41 protein polyclonal antibody (1:800) and goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (1:1000) (red). The pEGFP-C2 plasmid control was distributed throughout the cells (green). b DEF cells were co-transfected with pEGFP-C2-UL41 and pcDNA3.1-UL47. Much of the EGFP-UL41 fusion protein was localized to the nucleus (green), and pUL47 was mainly localized to the nucleus (red), detected with the rabbit anti-UL47 protein polyclonal antibody (1:1000) and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (1:1000). DEF cells were co-transfected with pEGFP-C2-UL41 and pcDNA3.1(+) as the control, and the EGFP-UL41 fusion protein was only distributed in the cytoplasm (green). c DEF cells were transfected with pcDNA3.1-UL47 Δ40–50 and pcDNA3.1-UL47 Δ768–777 , respectively. A portion of the ΔpUL47 was localized to the cytoplasm (green), detected with rabbit anti-UL47 protein polyclonal antibody (1:1000) and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (1:1000). d Representative western blotting results for DEF cells transfected with pEGFP-C2-UL41 (approximately 83 kDa) and pEGFP-C2 (approximately 27 kDa), detected with rabbit anti-EGFP antibody (1:2000) (Beyotime, Shanghai, China) and goat anti-rabbit IgG HRP-conjugated antibody (1:5000). Western blotting results for DEF cells transfected with pcDNA3.1(+), pcDNA3.1-UL47 Δ768–777 (approximately 87 kDa), pcDNA3.1-UL47 Δ40–50 (approximately 87 kDa) and pcDNA3.1-UL47 (approximately 89 kDa), respectively, detected with rabbit anti-UL47 antibody (1:1000) and goat anti-rabbit IgG HRP-conjugated antibody (1:5000). The β-actin antibody assessed β-actin as the loading control. M. Precision Plus Protein™ Dual Color Standards. e DEF cells were co-transfected with pEGFP-C2-UL41 and pcDNA3.1-UL47 Δ40–50 , pEGFP-C2-UL41 and pcDNA3.1-UL47 Δ768–777 , respectively. Much of the EGFP-UL41 fusion protein was localized to the cytoplasm (green). A portion of ΔpUL47 was localized to the cytoplasm (red), detected with rabbit anti-UL47 protein polyclonal antibody (1:1000) and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (1:1000)

Article Snippet: A portion of the ΔpUL47 was localized to the cytoplasm (green), detected with rabbit anti-UL47 protein polyclonal antibody (1:1000) and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (1:1000). d Representative western blotting results for DEF cells transfected with pEGFP-C2-UL41 (approximately 83 kDa) and pEGFP-C2 (approximately 27 kDa), detected with rabbit anti-EGFP antibody (1:2000) (Beyotime, Shanghai, China) and goat anti-rabbit IgG HRP-conjugated antibody (1:5000).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot