Journal: bioRxiv

Article Title: The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic

doi: 10.1101/2023.11.02.565271

Figure Lengend Snippet: Capture of AP-1 vesicles using magnetic beads. (A) A CCV-enriched fraction was prepared from HeLa cells expressing mRuby2-tagged AP-1 γ, then the AP-1-positive vesicles were captured just before the final centrifugation step using a rabbit antiserum against mRuby2 followed by protein A-coated magnetic beads. CCVs (arrowheads) can be seen to be associated with the beads. (B) Coomassie blue-stained gel of the whole cell homogenate, proteins captured on magnetic beads, and the CCV-enriched fraction from cells expressing mRuby2-tagged AP-1 γ. As a control for specificity, the preimmune serum from the same rabbit was also incubated with protein A beads. The samples containing beads and the CCV enriched-fraction were made up to the same volume so efficiency of capture could be assessed, and the sample containing cell homogenate was made up to the same protein concentration as the sample containing the CCV fraction. H and L refer to immunoglobulin heavy and light chains; the bands labelled 1-4 were excised and found to correspond to CIMPR, CHC17, the AP-1 γ subunit, and the AP-1 β1 subunit respectively. (C) Western blot of the samples shown in (B) probed with various antibodies. The dotted lines indicate the position on the gel for each antigen. In addition to the expected proteins, a small amount of AP-2 was also captured by the beads. GGA2 has a similar profile to AP-2, with relatively little captured by the beads.

Article Snippet: The following antibodies were used in this study: rabbit anti-CHC17 (one made in house and one purchased from Abcam, ab172958); mouse anti-CHC17 (X22, Abcam); rabbit anti-AP1G1 (made in house); rabbit anti-GFP (one a gift from Matthew Seaman, CIMR, Cambridge, and one purchased from Abcam, EPR14104); mouse anti-HaloTag (Promega, G9211); rabbit anti-CIMPR (made in house); mouse anti-AP-2 α subunits (AC1-M11, Abcam); and mouse anti-GGA2 (a gift from Doug Brooks, University of South Australia).

Techniques: Magnetic Beads, Expressing, Centrifugation, Staining, Incubation, Protein Concentration, Western Blot

Journal: bioRxiv

Article Title: The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic

doi: 10.1101/2023.11.02.565271

Figure Lengend Snippet: Proteomic analysis of the bead-captured samples. CCV-enriched fractions were prepared from HeLa cells expressing mRuby2-tagged AP-1 γ, grown in either SILAC heavy (H) or SILAC light (L) medium. AP-1-positive vesicles were captured from the H fraction before the final centrifugation step, while the L fraction was centrifuged to provide an input reference. Heavy-to-light ratios were calculated as a measure of enrichment in the bead-captured samples over the input and statistical analysis was performed for 632 proteins that passed quality control. (A) Data presented as a volcano plot. For each protein, a one-sample t -test was performed against a hypothetical ratio defined as the mean ratio for CLTC, the most abundant protein in both the H and L samples (see ). The proteins on the right side of the plot are potential components of AP-1 vesicles, with the most enriched hits being AP-1 subunits. The three hydrolase receptors and various hydrolases also score highly. Proteins on the left side are unlikely to be components of AP-1 vesicles, and they include ribosomal proteins. AP-2 subunits are generally to the left of clathrin, with the exception of AP2B1 (arrowhead), which is known to associate promiscuously with AP-1 . Other AP-1 cargo and accessory proteins are not indicated on this plot. (B) Protein identities in the boxed region in (A) , which contains the most enriched hits. The proteins shown in colour were also hits in our previous knocksideways analyses ( ; ). The arrowheads indicate the syntaxins STX6, STX10, and STX16. The complete dataset is available in Supplemental Table 1. Supplemental Table 2 contains proteomic data from unlabelled cells, in which the CCV-enriched fraction was incubated with beads coated with either anti-mRuby or preimmune serum, as further confirmation of the specificity of the AP-1 vesicle capture.

Article Snippet: The following antibodies were used in this study: rabbit anti-CHC17 (one made in house and one purchased from Abcam, ab172958); mouse anti-CHC17 (X22, Abcam); rabbit anti-AP1G1 (made in house); rabbit anti-GFP (one a gift from Matthew Seaman, CIMR, Cambridge, and one purchased from Abcam, EPR14104); mouse anti-HaloTag (Promega, G9211); rabbit anti-CIMPR (made in house); mouse anti-AP-2 α subunits (AC1-M11, Abcam); and mouse anti-GGA2 (a gift from Doug Brooks, University of South Australia).

Techniques: Expressing, Centrifugation, Incubation

Journal:

Article Title: Pax6 is essential for lens fiber cell differentiation

doi: 10.1242/dev.032888

Figure Lengend Snippet: Arrest of LFC differentiation following Pax6 loss is not mediated by upregulation of Sox2. (A) Sox2lox targeting vector and somatic deletion allele. The neo selection cassette is flanked by frt sites (blue triangles). The single Sox2 exon is flanked by loxP sites (green triangles). (B-M′) Cre-mediated deletion results in the Sox2Δ allele. Sox2lox/lox;Pax6lox/lox control (B-G) and Sox2lox/lox;Pax6lox/lox;Mlr10 E14.5 double somatic mutant (H-M′) mouse lenses analyzed by H&E staining (B,H) and antibody labeling for Sox2 and Ap2α (C,I, green and red, respectively), phosphohistone H3 (PH3, red in D,J,J′), αA-crystallin (E,K), β-crystallin (F,L) and cleaved caspase 3 (cCas, red in G,M,M′). Counterstaining was with DAPI (D,F,G,J,L,M). Arrows indicate the lens equator, arrowheads to the aberrant cells in the lens posterior. LE, lens epithelium; re, retina. Scale bar: 100 μm.

Article Snippet: Immunofluorescence analysis was performed on paraffin sections as previously described ( Ashery-Padan et al., 2000 ) using the following primary antibodies: rabbit anti-Pax6 (1:1000, Chemicon), mouse anti-Ap2α (1:50, Santa Cruz), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling), goat anti-αA-crystallin (1:1000, Santa Cruz), goat anti-αB-crystallin (1:100, Santa Cruz), rabbit anti-βB1-crystallin (1:250, Santa Cruz), rabbit anti-γF-crystallin (1:50, Santa Cruz), rabbit anti-cyclin D1 (1:250, Thermo Scientific), rat anti-Ki67 (1:100, Dako), goat anti-p57 Kip2 (1:100, Santa Cruz), rabbit anti-Prox1 (1:50, Acris) and rabbit anti-Sox2 (1:500, Chemicon).

Techniques: Plasmid Preparation, Selection, Mutagenesis, Staining, Antibody Labeling

Journal:

Article Title: Pax6 is essential for lens fiber cell differentiation

doi: 10.1242/dev.032888

Figure Lengend Snippet: Pax6 downregulates Sox2 in the lens equator. (A,B) Immunofluorescent detection of Sox2 (green) and Ap2α (red) in E14.5 control (A) and Pax6lox/lox;Mlr10 (B) mouse lenses. Arrowheads in B indicate elevated expression of Sox2 at the lens equator and in the posterior lens. (A′,B′) Counterstaining of A,B with DAPI. (C) Quantification of Sox2 protein by confocal image analysis (n=6, **P<0.001). eq, lens equator; LE, lens epithelium; re, retina. Scale bar: 100 μm.

Article Snippet: Immunofluorescence analysis was performed on paraffin sections as previously described ( Ashery-Padan et al., 2000 ) using the following primary antibodies: rabbit anti-Pax6 (1:1000, Chemicon), mouse anti-Ap2α (1:50, Santa Cruz), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling), goat anti-αA-crystallin (1:1000, Santa Cruz), goat anti-αB-crystallin (1:100, Santa Cruz), rabbit anti-βB1-crystallin (1:250, Santa Cruz), rabbit anti-γF-crystallin (1:50, Santa Cruz), rabbit anti-cyclin D1 (1:250, Thermo Scientific), rat anti-Ki67 (1:100, Dako), goat anti-p57 Kip2 (1:100, Santa Cruz), rabbit anti-Prox1 (1:50, Acris) and rabbit anti-Sox2 (1:500, Chemicon).

Techniques: Expressing

Journal: Protein Engineering, Design and Selection

Article Title: A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes

doi: 10.1093/protein/gzz035

Figure Lengend Snippet: Capture and detection of TfR:AP-2 complexes on the yeast surface. (A) Schematic representation of the fYDIP concept. Plasma membrane protein complexes from cell-surface biotinylated HEK293 cells are solubilized and incubated with yeast displaying anti-TfR scFv, H7, for 2 hours to capture TfR:AP-2 complexes (i). Subsequently, captured complexes are labeled with either (ii) streptavidin-PE (SA-PE) to detect the presence of biotinylated TfR or (iii) anti-c-myc to detect full-length scFv expression and anti-AP2α to detect TfR-associated AP-2. Yeasts displaying D1.3 scFv are used as a negative control. (B) Flow cytometry histograms (TfR binding detected via streptavidin-PE) demonstrate the capture of TfR by H7 displaying yeast. (C) Flow cytometry dot plots (x-axis detection with anti-AP2α antibody and y-axis detection with anti-c-myc antibody) demonstrate the presence of measurable levels of AP-2 in the H7 captured complexes. Inset numbers in the upper two quadrants indicate the percentage of the scFv-displaying yeast population found in each quadrant. (D) Western blot of complexes eluted from the yeast surface. Blots were probed for the presence of TfR (Anti-TfR Ab, ~ 95 kDa) and AP-2 (Anti-AP2α Ab, ~ 100 kDa).

Article Snippet: Binding to complexes containing biotinylated membrane proteins was detected with streptavidin R-Phycoerythrin conjugate (SA10041, Thermo Fisher), and association with AP-2 was detected via labeling with mouse-anti-AP2α (Clone AP.6, Thermo Fisher) followed by goat-anti-mouse IgG-allophycocyanin.

Techniques: Incubation, Labeling, Expressing, Negative Control, Flow Cytometry, Binding Assay, Western Blot

Journal: Protein Engineering, Design and Selection

Article Title: A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes

doi: 10.1093/protein/gzz035

Figure Lengend Snippet: Antibody library screening via fYDIP. (A) Analytical flow cytometry dot plots demonstrate enrichment of scFv binding to complexes containing biotinylated plasma membrane proteins over two rounds of screening. scFv expression is depicted on the x-axis using anti-c-myc antibody for detection, and biotinylated BMPM binding is depicted on the y-axis using an anti-biotin antibody for detection. The percentage of scFv-expressing cells falling in the double positive gate is displayed in the upper right of each plot. (B) Analytical flow cytometry dot plots demonstrate the enrichment of clones that bind to complexes containing both biotinylated BMPM proteins and AP-2 after one additional round of functional screening. Biotinylated BMPM binding is depicted on the y-axis using streptavidin detection, and AP-2 association is depicted on the x-axis using an anti-AP2α antibody for detection. The percentage of BMPM binding clones that fall in the double positive gate is displayed.

Article Snippet: Binding to complexes containing biotinylated membrane proteins was detected with streptavidin R-Phycoerythrin conjugate (SA10041, Thermo Fisher), and association with AP-2 was detected via labeling with mouse-anti-AP2α (Clone AP.6, Thermo Fisher) followed by goat-anti-mouse IgG-allophycocyanin.

Techniques: Library Screening, Flow Cytometry, Binding Assay, Expressing, Clone Assay, Functional Assay

Journal: Protein Engineering, Design and Selection

Article Title: A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes

doi: 10.1093/protein/gzz035

Figure Lengend Snippet: Individual clone analysis by fYDIP. Three independent transformants of the indicated monoclonal yeast were incubated with rat BMPMs from a single microvessel isolation and analyzed via flow cytometry (A–C). (A) Representative flow cytometry histograms demonstrate binding to complexes containing biotinylated BMPM proteins through detection with an anti-biotin antibody. Clone D1.3 binds to hen egg lysozyme and is included as a nonbinding control. (B) Representative flow cytometry dot plots for the assessment of AP-2 association in the bound complexes. scFv expression is depicted on the y-axis, and AP-2 association is depicted on the x-axis as described in Fig. 3. Inset percentages in the upper two quadrants of each plot represent the percentage of scFv-expressing cells detected in that quadrant. (C) Quantification of the geometric mean fluorescence intensity of the BMPM binding signal (white bars, left axis) and AP-2 association signal (black bars, right axis) for each monoclonal scFv in (A) and (B). Data are the mean of n = 3 biological replicates, and error bars represent standard deviation. Significance of BMPM binding and AP-2 association signal above background was assessed using unpaired Student’s t-tests comparing each individual clone with the negative control D1.3 background signal intensity. ns = not significant, **P < 0.001, ***P < 0.0001. (D) fYDIP eluates for the indicated monoclonal scFv were analyzed via anti-AP2α western blot to confirm the presence of AP-2 in the captured complexes. The AP-2α subunit was detected at the expected molecular weight of ~ 100 kDa.

Article Snippet: Binding to complexes containing biotinylated membrane proteins was detected with streptavidin R-Phycoerythrin conjugate (SA10041, Thermo Fisher), and association with AP-2 was detected via labeling with mouse-anti-AP2α (Clone AP.6, Thermo Fisher) followed by goat-anti-mouse IgG-allophycocyanin.

Techniques: Incubation, Isolation, Flow Cytometry, Binding Assay, Expressing, Fluorescence, Standard Deviation, Negative Control, Western Blot, Molecular Weight

Journal: Protein Engineering, Design and Selection

Article Title: A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes

doi: 10.1093/protein/gzz035

Figure Lengend Snippet: Immunoprecipitation of antigen complexes for mass spectrometry. (A) Eluates from immunoprecipitation and raw RBE4 lysate were analyzed by anti-AP2α western blot. (B) Eluates from immunoprecipitation were also analyzed by Coomassie blue stained SDS-PAGE gel. The indicated bands (* and **) were excised, digested with trypsin and analyzed via LC-MS/MS. * Specific band identified as Myh9, ** Specific band identified as AHNAK.

Article Snippet: Binding to complexes containing biotinylated membrane proteins was detected with streptavidin R-Phycoerythrin conjugate (SA10041, Thermo Fisher), and association with AP-2 was detected via labeling with mouse-anti-AP2α (Clone AP.6, Thermo Fisher) followed by goat-anti-mouse IgG-allophycocyanin.

Techniques: Immunoprecipitation, Mass Spectrometry, Western Blot, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Reports Medicine

Article Title: Lhx2 promotes axon regeneration of adult retinal ganglion cells and rescues neurodegeneration in mouse models of glaucoma

doi: 10.1016/j.xcrm.2024.101554

Figure Lengend Snippet: Lhx2- mediated axon regeneration is specific to RGCs (A) Timeline of the experiment. (B) Schematic of the retinal structure of Vglut2-Cre mouse expressing Lhx2 in RGCs specifically. (C) Confocal image showing that Lhx2 (red) was specifically expressed in RBPMS+ (green+) and AP2α− (white−) labeled RGCs. (D) Axon regeneration was analyzed by CTB-555 tracing. Left, confocal images showing that Lhx2 expression in RGCs induced axon regeneration 14 days after optic nerve crush in Vglut2-cre mouse. Right, the columns display magnified images of the areas in the white dashed rectangles at 0.5, 1.0, 1.75, and 2.0 mm distal to the crush sites. Scale bar, 40 mm. (E) Quantification of regenerating axons at different distances distal to the nerve crush site (0.25–2.5 mm) (data are presented as mean ± SEM; one-way ANOVA followed by Tukey’s multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; AAV2-Flex-EGFP: n = 3 mice, AAV2-Flex-LHX2: n = 3 mice). (F) Schematic of retinal structure showing Vgat2-cre mouse expressing Lhx2 in inhibitory neurons specifically. (G) Confocal image of RBPMS− (green−) and AP2α+ (white+) labeled inhibitory neuron (amacrine cells) expressing Lhx2 (red). (H) Axon regeneration was analyzed by CTB-555 tracing. Confocal images showing that Lhx2 overexpression in inhibitory neuron did not induce axon regeneration 14 days after optic nerve crush in Vgat2-cre mouse. Scale bar, 250 μm. (I) Quantification of regenerating axons at different distances distal to the nerve crush site (0.25–1.0 mm) (data are presented as mean ± SEM; one-way ANOVA followed by Tukey’s multiple comparisons test; ns, not significant; AAV2-Flex-EGFP: n = 5 mice, AAV2-Flex-LHX2: n = 5 mice).

Article Snippet: Primary antibodies: Mouse anti-Tuj1 (1:1,000, BioLegend, 801202), Rabbit anti-Lhx2 (1:500, Abcam, ab184337), Rabbit anti-RBPMS (1:500, Abcam, ab194213), Guinea pig anti-RBPMS (1:200, PhosphoSolutions, 1832-RBPMS), Mouse anti-AP2α (1:50, DSHB, AB_528084), Rabbit anti-Sema3C (1:200, Solaibao, K010031P), Rabbit anti-Pmp22 (1:200, Solaibao, K107046P).

Techniques: Expressing, Labeling, Over Expression

Journal: Cell Reports Medicine

Article Title: Lhx2 promotes axon regeneration of adult retinal ganglion cells and rescues neurodegeneration in mouse models of glaucoma

doi: 10.1016/j.xcrm.2024.101554

Figure Lengend Snippet:

Article Snippet: Primary antibodies: Mouse anti-Tuj1 (1:1,000, BioLegend, 801202), Rabbit anti-Lhx2 (1:500, Abcam, ab184337), Rabbit anti-RBPMS (1:500, Abcam, ab194213), Guinea pig anti-RBPMS (1:200, PhosphoSolutions, 1832-RBPMS), Mouse anti-AP2α (1:50, DSHB, AB_528084), Rabbit anti-Sema3C (1:200, Solaibao, K010031P), Rabbit anti-Pmp22 (1:200, Solaibao, K107046P).

Techniques: Recombinant, DNA Extraction, Sequencing, Software