Journal: Investigative Ophthalmology & Visual Science

Article Title: Proliferator–Activated Receptor Alpha Inhibits Abnormal Extracellular Matrix Accumulation and Maintains Energy Metabolism in Late-Onset Fuchs Endothelial Corneal Dystrophy

doi: 10.1167/iovs.66.4.36

Figure Lengend Snippet: Primary Antibodies Used in Immunofluorescence

Article Snippet: Rat , ZO1 , 1:100 , Santa Cruz , USA.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: A vertebrate Vangl2 translational variant required for planar cell polarity

doi: 10.1016/j.jbc.2024.106792

Figure Lengend Snippet: Identification of VANGL2-Long, a highly conserved N terminally extended VANGL2 isoform, in human and mouse cells. A , Western blot analysis of murine epithelial (IMCD3) and human embryonic kidney (HEK 293T) cell extracts using VANGL2-specific mAb 36E3 ( top panel ). Arrows point to the major 62 Kd VANGL2 protein and to a less intense and slower migrating band of 70 Kd both recognized by mAb 36E3. Immunoblot with an anti-alpha tubulin ( bottom panel ) was used as a loading control. B , DNA sequence of the 5′ region of human VANGL2 cDNA encompassing 342 bp of the 5′-UTR region immediately upstream to the conventional ATG start site (highlighted in red ) and the first 57 bp of ATG-initiated VANGL2 ORF. A potential ATA alternative initiation site in position −144 (ATA -144 ) relative to the canonical VANGL2 ATG 1 start site is highlighted in magenta . Amino acid sequences of VANGL2 and the N-terminal extension of VANGL2-Long are highlighted in yellow and cyan , respectively. The sequence of the peptide used to generate the N-VGL2 pAb rabbit antibodies is indicated in bold letters . C , diagram representations of the human VANGL2 mRNA ( top ) with its canonical (AUG) and near-cognate (ATA -144 ) initiation sites and the two VANGL2 ( middle ) and VANGL2-Long ( bottom ) encoded isoforms. Met-initiated VANGL2 and the ATA-initiated N-terminal extension are drawn in yellow and cyan , respectively. Black rectangles represent the four transmembrane domains present in both isoforms and numbers on the right give their respective length. The sequence of a peptide identified by mass spectrometry and encompassing the N terminus of VANGL2 ( black ) preceded by three residues of the 5′-UTR region ( cyan ) is shown below the VANGL2-Long diagram. Brackets above the two schematized isoforms indicate a common region recognized by mAb 36E3. D , schemes of pVANGL2 ( top ), pVANGL2-SL ( middle ), and pVANGL2-Long ( bottom ) vectors. All VANGL2 constructs are under the control of CMV promoter ( gray box ) and VANGL2 isoform representation uses the same color code as in ( C ). Note that, as indicated in red in pVangl2-Long, ATA -144 and the canonical ATG codon have been mutated to, respectively, ATG and GCG in order to enhance VANGL2-Long biosynthesis while abrogating VANGL2 expression by this construct. E , HEK 293T ( left panel ) and IMCD3 ( right panel ) cells were probed by immunoblotting with anti-VANGL2 mAb 36E3 upon transfection with the indicated plasmids. Note that the absence of endogenous VANGL2 signals in HEK 293T cells transfected with an empty plasmid (vector) is due to the short exposure time used to detect the overexpressed VANGL2 isoforms. Arrows point to VANGL2 and VANGL2-Long isoforms that are overexpressed by the relevant pVANGL2 and pVANGL2-Long constructs. Note that pVANGL2 SL promotes the synthesis of both VANGL2 and VANGL2-Long simultaneously ( left panel ). F , VANGL2-L expression requires a near-cognate AUA alternative initiation site in position −144. HEK293T cells transfected with either an empty plasmid (vector), p VANGL2, or pVANGL2-SL plasmids in which the predicted alternative AUA-144 start codon was either left intact (AUA-144) or mutated to GCA (GCA-144) were processed for Western blot analysis with mAb36E3 to detect all VANGL2 isoforms. G , amino acid sequence alignment of predicted N-terminal extensions from six vertebrate species, including human ( Hs ), mouse ( Ms ), chicken ( Gg ), python ( Pb ), African clawed frog ( Xl ), and zebrafish ( Dr ). Strictly conserved residues are in bold letters . AUA-encoded isoleucine residues that initiate the translation of all the extensions shown here are highlighted in magenta , whereas the AUG-encoded methionine residue of Vangl2 is in red . H , proteins extracted from SKBR7 ( left panel ) and IMCD3 ( right panel ) cells were immunoprecipitated with the VANGL2-Long isoform-specific N-Vgl2 pAbs and immunopurified proteins were immunoblotted with mAb 36E3. Control Ab is an isotypic control rabbit antibody. pAb, polyclonal antibody.

Article Snippet: Incubation with the primary antibodies were done O/N at 4 °C at the following concentrations: chicken anti-GFP (Aves GFP-1020, 1/500), rabbit anti-RFP (600401379 Rockland, 1:500), rat anti-Vgl2 (36E3) homemade (1:5000), mouse anti-ZO1-TJP1 (Thermo Fisher Scientific 33-9100, 1:200), mouse anti-γ-tubulin (Abcam Ab 11316, 1:800), mouse anti-acetylated-tubulin (Sigma T7451, 1/1000), and mouse anti-Centrin (Millipore cat#04-1624, 1:500).

Techniques: Western Blot, Control, Sequencing, Mass Spectrometry, Construct, Expressing, Transfection, Plasmid Preparation, Residue, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: A vertebrate Vangl2 translational variant required for planar cell polarity

doi: 10.1016/j.jbc.2024.106792

Figure Lengend Snippet: VANGL2 and VANGL2-Long form a tripartite complex with Vangl1 in vivo . A , immunoprecipitation of lysates from HEK 293T cells transfected with GFP-VANGL1 and a control plasmid or GFP-VANGL1 in combination with pVANGL2 or pVANGL2-Long, using mAb 36E3 or N-VGL2 pAbs, as indicated. The presence of GFP-VANGL1 and VANGL2 in the immunoprecipitated samples was determined by Western blotting using, respectively, a GFP-specific antibody ( bottom panels ) and mAb 36E3 ( top panels ). Arrows point to the VANGL2 and VANGL2-Long signals detected by mAb 36E3. B , SKBR7 cell extracts were immunoprecipitated with either mAb 36E3 ( left panels ) or N-VGL2 pAb ( right panels ), followed by a Western blot analysis of VANGL1 ( bottom panels ) and VANGL2 isoforms ( top panels ) using, respectively, mAb 19D5 and mAb 36E3 on two different membranes. Isotopic rat and rabbit antibodies were used as negative controls for each IP experiment (Ctrl Ab). C , lysates from WT and Crispr-Cas9 KO- VANGL2 (KO) HEK 293T cells were first immunoprecipitated with N-VGL2 pAb (IP1, N-VGL2 Ab). Bound proteins were subsequently eluted with the relevant immunogenic peptide (eluate) before being subjected to a second round of IP with VANGL1-specific mAb 19D5 (IP2, α-VANGL1). The different fractions were examined by immunoblotting for their VANGL1 ( lower panel ) and VANGL2 ( upper panel ) contents using, respectively, mAb 19D5 and mAb 36E3. Arrows in the upper panel indicate the two endogenous VANGL2 and VANGL2-Long isoforms, while those in the lower panel point to Vangl1 and immunoglobulin heavy chains (Ig HC ). pAb, polyclonal antibody.

Article Snippet: Incubation with the primary antibodies were done O/N at 4 °C at the following concentrations: chicken anti-GFP (Aves GFP-1020, 1/500), rabbit anti-RFP (600401379 Rockland, 1:500), rat anti-Vgl2 (36E3) homemade (1:5000), mouse anti-ZO1-TJP1 (Thermo Fisher Scientific 33-9100, 1:200), mouse anti-γ-tubulin (Abcam Ab 11316, 1:800), mouse anti-acetylated-tubulin (Sigma T7451, 1/1000), and mouse anti-Centrin (Millipore cat#04-1624, 1:500).

Techniques: In Vivo, Immunoprecipitation, Transfection, Control, Plasmid Preparation, Western Blot, CRISPR

Journal: The Journal of Biological Chemistry

Article Title: A vertebrate Vangl2 translational variant required for planar cell polarity

doi: 10.1016/j.jbc.2024.106792

Figure Lengend Snippet: xVangl2-Long expression during Xenopus embryogenesis. A , whole Xenopus embryos collected at the indicated stages of development were used for protein extraction and Western blot analysis with mAb 36E3 ( upper panel ). Anti-tubulin immunoblotting was used to control protein loading ( lower panel ). The bands corresponding to xVangl2 and xVangl2-Long are indicated by black arrowheads . B , graphical representation of xVangl2 and xVangl2-Long normalized levels corresponding to the blot shown in ( A ). C , embryos at stage 14 were dissected to isolate the neural plate from the rest of the embryo, which comprised endodermal, ectodermal, and mesodermal tissues (End + Ect + Mes). Proteins were extracted and probed by Western blotting with mAb 36E3 ( upper panel ) and anti-tubulin ( lower panel ). D and E , spatial distribution of Vangl2 proteins revealed by immunofluorescence (IF) with mAb 36E3 in neural plate cells at stage 16 ( D ) and in epidermis at stage 31 ( E ). ZO1 IF was used to delineate cell junctions. White arrowheads point to polarized Vangl2 enrichment in the plasma membrane, which is anterior in neural plate cells and posterior in epidermal cells. F , GFP-xVangl2 and GFP-xVangl2-Long synthetic mRNAs were injected in dorsal blastomeres at 4-cell stage to reveal the spatial distribution of either Vangl2 isoform in neural plate cells of live stage 16 embryos. mRFP mRNA coinjection was used to delineate cell contours. White arrowheads point to anterior enrichment of Vangl2 and Vangl2-Long. In all panels, the scale bars represent 10 μm. MBT, midblastula transition.

Article Snippet: Incubation with the primary antibodies were done O/N at 4 °C at the following concentrations: chicken anti-GFP (Aves GFP-1020, 1/500), rabbit anti-RFP (600401379 Rockland, 1:500), rat anti-Vgl2 (36E3) homemade (1:5000), mouse anti-ZO1-TJP1 (Thermo Fisher Scientific 33-9100, 1:200), mouse anti-γ-tubulin (Abcam Ab 11316, 1:800), mouse anti-acetylated-tubulin (Sigma T7451, 1/1000), and mouse anti-Centrin (Millipore cat#04-1624, 1:500).

Techniques: Expressing, Protein Extraction, Western Blot, Control, Immunofluorescence, Clinical Proteomics, Membrane, Injection

Journal: Frontiers in Pharmacology

Article Title: Usnea improves high-fat diet- and vitamin D3-induced atherosclerosis in rats by remodeling intestinal flora homeostasis

doi: 10.3389/fphar.2022.1064872

Figure Lengend Snippet: UEE affected the concentrations of occludin and ZO-1. (A) UEE increased the expression of ZO-1; (B) UEE increased the concentration of occludin; and (C) potential correlation analysis between the intestinal flora associated with AS and pharmacokinetic markers. Rectangles represent the intestinal flora related to AS; circles denote pharmacokinetic markers; red indicates a positive association, whereas blue indicates a negative correlation. Results are expressed as the mean ± SD ( n = 3 per group). * p < 0.05, ** p < 0.01.

Article Snippet: Anti-CYP7A1 rabbit monoclonal antibody (ID: 510615), anti-zonula occludens-1 (ZO-1) rabbit monoclonal antibody (ID: AF5145), anti-occludin rabbit monoclonal antibody (ID: 502601), anti-FMO3 rabbit monoclonal antibody (ID: 389347), anti-β-actin rat antibody (ID: DD1027), goat anti-rabbit IgG (H&L) (ID: JJ0903), and goat anti-rat secondary antibody (ID: II1120) were provided by Chengdu Zen Bioscience Co., Ltd. (Chengdu, China).

Techniques: Expressing, Concentration Assay