Journal: Current protocols in protein science

Article Title: Protein Detection in Gels Using Fixation UNIT 10.5

doi: 10.1002/cpps.48

Figure Lengend Snippet: Overview of Common Gel Staining Methods

Article Snippet: Representative Common Coomassie Stains table ft1 table-wrap mode="anchored" t5 Table 10.5.4 caption a7 Representative Vendors Stain Limit of Detection Time MS Compatible BioRad Flamingo Fluorescent Gel Stain 0.25–0.5 ng 5 hr yes Oriole Fluorescent Gel Stain 0.5–1 ng 1.5 hr Thermo Fisher CoomassieTM Fluor Orange 8 ng 1 hr yes Krypton Fluorescent Protein Stain 0.25 ng 2 hr 40 min Thermo Fisher; Sigma; BioRad; Lonza SYPRO ® Ruby 0.25 ng 90 min a /18 hr yes Thermo Fisher; Sigma SYPRO ® Orange 4–8 ng ~1 hr yes Thermo Fisher; Lonza SYPRO ® Red 4–8 ng ~1 hr yes SYPRO ® Tangerine 4 ng ~1 hr yes Open in a separate window a The 90 min rapid protocol requires microwaving the gel and staining solution for 3 × 30 sec, or alternatively incubating the gel at 80°C for 30 min followed by subsequent destaining.

Techniques: Staining, Quantitation Assay, Mass Spectrometry, Silver Staining

Journal: Current protocols in protein science

Article Title: Protein Detection in Gels Using Fixation UNIT 10.5

doi: 10.1002/cpps.48

Figure Lengend Snippet: Representative Common Fluorescent Stains

Article Snippet: Representative Common Coomassie Stains table ft1 table-wrap mode="anchored" t5 Table 10.5.4 caption a7 Representative Vendors Stain Limit of Detection Time MS Compatible BioRad Flamingo Fluorescent Gel Stain 0.25–0.5 ng 5 hr yes Oriole Fluorescent Gel Stain 0.5–1 ng 1.5 hr Thermo Fisher CoomassieTM Fluor Orange 8 ng 1 hr yes Krypton Fluorescent Protein Stain 0.25 ng 2 hr 40 min Thermo Fisher; Sigma; BioRad; Lonza SYPRO ® Ruby 0.25 ng 90 min a /18 hr yes Thermo Fisher; Sigma SYPRO ® Orange 4–8 ng ~1 hr yes Thermo Fisher; Lonza SYPRO ® Red 4–8 ng ~1 hr yes SYPRO ® Tangerine 4 ng ~1 hr yes Open in a separate window a The 90 min rapid protocol requires microwaving the gel and staining solution for 3 × 30 sec, or alternatively incubating the gel at 80°C for 30 min followed by subsequent destaining.

Techniques: Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Generation of Celsr1 and Celsr2 loss-of function mutant mice by CRISPR/Cas9. (A) Schematic representation of Celsr1 and Celsr2 protein domains. The two proteins are 55% identical in amino acid sequence and have the same overall domain organization. (B) CRISPR-Cas9 targeting of Celsr1 and Celsr2 genomic loci. Guide RNAs were targeted to the sequence encoding the signal peptide for each of Celsr1 and Celsr2 . The resulting targeted alleles are shown with the ATG and signal sequence in purple font and deleted sequences highlighted in yellow. (C) Celsr1 −/− and wild type ( WT ) littermate at P12. Note curly tail and whorled hair pattern on the head of Celsr1 −/− homozygote. (D) Left and right paws of Celsr1 −/− and WT littermate at P12. Celsr1 −/− homozygotes exhibit prominent hair whorl on each paw. (E) Celsr1 −/− and WT littermate embryos at E15.5. Celsr1 −/− homozygotes display curly tail. (F) Western blot of epidermal lysates from WT and Celsr1 −/− P0-P3 backskins with anti-Celsr1 antibody. (G) Western blot of epidermal lysates from three individual WT and two individual Celsr2 −/− P0-P3 pups with anti-Celsr2 antibody. (H) Confocal immunofluorescence image of whole mount epidermis from E15.5 WT and Celsr1 −/− mutant embryos labeled with Celsr1 antibodies. Scale bars: 10 µm. (I) Quantification of Celsr1 mean fluorescence intensity in WT and Celsr1 −/− mutant epidermis (n = 3 skin regions from 4 different WT embryos and n = 3 skin regions from 3 different Celsr1 −/− embryos).

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Mutagenesis, CRISPR, Sequencing, Western Blot, Immunofluorescence, Labeling, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Celsr1, but not Celsr2, is necessary for correct asymmetric orientation of developing hair follicles. (A) Average intensity projection of WT embryonic back skin at E15.5, labelled for P-cadherin (green) and Sox9 (magenta). White box denotes zoomed in region shown below, left. Average intensity projection of a typical WT hair follicle imaged at higher mag (below, right). Scale bars: 1000, 200, and 25 µm, respectively. Anterior is to the left. (B–D) As for (A) , except Celsr1 −/− , Celsr2 −/− and Celsr1 −/− ;Celsr2 −/− respectively. (E) Bar chart showing cumulative percentage of polarized (grey bar) vs. non-polarized (white bar) hair follicles in n = 3 E15.5 back skins from 3 different embryos. n in figure represents total number of follicles analyzed. Error bars = SEM. (F–H) As for (E) , except Celsr1 −/− , Celsr2 −/− and Celsr1 −/− ;Celsr2 −/− respectively. (I) Rose plot of polarized follicles in (E) showing the angle of orientation, with anterior = 0° and posterior = 180°. Shaded areas in bars represent relative contribution of each replicate (n = 3 backskins from 3 different embryos), with n in figure representing total number of polarized hair follicles analyzed. (J–L) As for (I) , except Celsr1 −/− , Celsr2 −/− and Celsr1 −/− ;Celsr2 −/− respectively.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Loss of core PCP protein asymmetry in the epidermis of Celsr1 −/− and Celsr1 −/− ; Celsr2 −/− double mutants. (A–H) Representative planar views of the basal layer of the interfollicular epidermis at E15.5 showing Celsr1, Fz6 and Vangl2 distribution as detected by immunofluorescence. Anterior is to the left. Scale bar: 20 µm. Magnified areas below are overlaid with colored lines representing the axis (line angle) and magnitude (line length) of polarity. Quantification of polarity distributions are displayed below on circular histograms. (A-B′) Celsr1 (A) , Fz6 (B) and Vangl2 (B′) in WT embryos, n = 11,951 basal cells, 3 embryos. (C-D′) Celsr1 (C) , Fz6 (D) , and Vangl2 (D′) in Celsr1 −/− embryos, n = 11,629 basal cells, 3 embryos. (E-F′) Celsr1 (E) , Fz6 (F) and Vangl2 (F′) in Celsr2 −/− embryos, n = 12,099 basal cells, 3 embryos. (G-H′) Celsr1 (G) , Fz6 (H) and Vangl2 (H′) in Celsr1 −/− ; Celsr2 −/− embryos, n = 9,064 basal cells, 3 embryos.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Celsr2 enriches at cell-cell junctions by homotypic interactions less efficiently than Celsr1. (A) Representative images of cell pairs expressing Celsr1-GFP, Celsr2-GFP or GFP-CAAX as indicated. Bottom panels show zoomed in junctional regions. Note the stronger enrichment of Celsr1-GFP at junctions compared to Celsr2-GFP and both isoforms are significantly more enriched at junctions compared to a non-junctional plasma membrane marker GFP-CAAX. Scale bars 20 µm (top panel) and 10 µm (bottom panel). (B) Plot of the junctional enrichment score (ratio of junctional mean intensity to the mean intensity of the cell pair). n = 32 Celsr1-GFP junctions, n = 43 Celsr2-GFP junctions, n = 60 GFP-CAAX junctions. Kolmogorov-Smirnov test p < 0.0001. Data pooled from two independent experiments where each experiment reflects the represented trend. (C) Fluorescence Recovery After Photobleaching (FRAP) of junctional Celsr1-GFP and Celsr2-GFP. Shown are representative images of the junctional region between cell pairs expressing Celsr1-GFP or Celsr2-GFP before and after bleaching as indicated. Bleached ROIs are marked by yellow arrowheads. (D) FRAP recovery plots. Shown is the normalized mean intensity with standard deviations of the bleach and recovery profiles plotted versus time for Celsr1-GFP (blue) and Celsr2-GFP (magenta) at junctions (in bold) and free cell edges that are not juxtaposed to a transfected cell (in lighter shade). (n = 36 ROIs for Celsr1 edge, 38 ROIs for Celsr2 edge, 78 ROIs for Celsr1 junctions and 75 ROIs for Celsr2 junctions). Data pooled from two independent experiments for cell edge measurements and three independent experiments for junction measurements. (E) Cell mixing experiment between cells expressing Celsr1-3xFLAG and Celsr2-GFP (top panels) or Celsr1-3xFLAG and Celsr1-GFP (bottom panels). Images show cell pairs forming heterotypic junctions. Celsr1-3xFLAG appears to enrich with both Celsr1-GFP and Celsr2-GFP, in trans , across cell-cell junctions. (F) Histogram depicting the frequency of Celsr1-3xFLAG: Celsr1-GFP and Celsr1-3xFLAG::Celsr2-GFP junctions across the range of junction enrichment ratios obtained for Celsr1-GFP and Celsr2-GFP, respectively. Inset shows box plot for the junction enrichment values of Celsr1-GFP and Celsr2-GFP. n = 56 Celsr1-GFP junctions and n = 64 Celsr2-GFP junctions. Kolmogorov-Smirnov test, p = 0.0004. Data pooled from two independent experiments.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Expressing, Clinical Proteomics, Membrane, Marker, Fluorescence, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Celsr2 recruits Fz6 and Vangl2 to keratinocyte junctions, similar to Celsr1. (A–C) Representative cell pair co-expressing Celsr1-GFP and Fz6-tdTomato (A) , tdTomato-Vangl2 (B) or a non-junctional membrane marker tdTomato-CAAX (C) . Arrowheads mark the junction between 2 cells and a magnified view of the junction is represented below the respective images. Scale bars = 20um. (D–F) Representative cell pair co-expressing Celsr2-GFP and Fz6-tdTomato (D) , tdTomato-Vangl2 (E) and tdTomato-CAAX (F) . Arrowheads mark the junction between 2 cells and a magnified view of the junction is represented below the respective images. Scale bars = 20 um. (G) Box plots depicting junction enrichment ratios for Fz6-tdTomato compared to tdTomato-CAAX when co-expressed with Celsr1-GFP or Celsr2-GFP (n = 33 for Celsr1-tdTomato CAAX, n = 49 for Celsr2-tdTomato-CAAX, n = 36 for Celsr1-Fz6-tdTomato, n = 66 for Celsr1-Fz6-tdTomato). (H) Box plots depicting junction enrichment values of Celsr1-GFP versus Celsr2-GFP in cells co-expressing Fz6-tdTomato. (I) Box plots depicting junction enrichment ratios for tdTomato-Vangl2 compared to tdTomato-CAAX when co-expressed with Celsr1-GFP or Celsr2-GFP (n = 66- Celsr1-tdTomatoCAAX, n = 87-Celsr2 tdTomatoCAAX, n = 63-Celsr1 tdTomatoCAAX, n = 67-Celsr2-tdTomatoCAAX). (J) Box plots depicting junction enrichment of Celsr1-GFP and Celsr2-GFP in cells expressing tdTomato-Vangl2. Data pooled from two independent experiments for Fz6-tdTomato and three independent experiments for tdTomato-Vangl2. Kolmogorov-Smirnov tests, **** p < 0.0001, ** p = 0.009.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Expressing, Membrane, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Product information of key antibodies and reagents used in this study.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Knock-Out

Journal: Frontiers in Cell and Developmental Biology

Article Title: Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity

doi: 10.3389/fcell.2022.1064907

Figure Lengend Snippet: Genotyping details for Celsr1 and Celsr2 including primer sequences and product sizes for WT and knockout animals.

Article Snippet: Standard protocols were performed for western blot- proteins were resolved on a 7.5% SDS gel, transferred to a nitrocellulose membrane (Bio-Rad), and detected using primary antibodies against Celsr1 , Celsr2 (goat, R&D Systems, 1:200), and E-cadherin (rabbit, Cell Signaling, 1:250 or rat, ThermoFisher, 1:1000).

Techniques: Knock-Out, Western Blot, Plasmid Preparation, Software