243 recombinant human cd31 protein  (Servicebio Inc)

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 2. Cytotoxicity, Oligomer Formation, and Lethal Effects of CPB Depend on CD31 Expression (A) Viability of bEndwt, bEndCD31ko, and bEnd- CD31ko transduced with CD31-GFP or CD54-GFP incubated with indicated doses of CPB (24 h, 37C) as a percentage of untreated control cells. See also Figure S2A. (B) Viability of WT EpH4 and EpH4 cells stably expressing CD31-GFP or CD54-GFP incubated as in (A). Data in (A and B) are represented as means (N = 8) ± SD. Multiple comparison two-way ANOVA, Sidak’s multiple comparison test, values with p < 0.0001 are indicated by an asterisk. (C) Western blots of EpH4, bEndwt, and bEnd- CD31ko incubated with 8 mg/mL CPB for 30 min at 37C and probed with indicated antibodies. (D) Effect of CD31 depletion on the lethal activity of CPB in C57BL/6 mice. WT and CD31-knockout mice were injected (i.p.) with 1 mg CPB per 20 g body weight. As a control, WT mice were injected with neutralized CPB. WT mice (n = 16), CD31- knockout mice (n = 17), and control (n = 13). Log- rank test, * significance (p < 0.0001). See also Figure S3A.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Expressing, Transduction, Incubation, Control, Stable Transfection, Comparison, Western Blot, Activity Assay, Knock-Out, Injection

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 3. CPB Targets CD31 to Kill Endothelial Cells (A) Immunofluorescence micrographs of bEndwt cells incubated in the presence and absence of 1 mg/mL CPB (10 min, 37C). CPB (green), CD31 (red), DNA (blue). Scale bar, 30 mm. (B) Left panel: maximum intensity projections of z stacks of representative micrographs of in situ PLA of bEndwt and bEndCD31ko cells incubated in the presence and absence of 1 mg/mL CPB (10 min, 37C). PLA signal (red), DNA (blue). Scale bar, 60 mm. Right panel: Box plots of quantification of PLA puncta per cell type. ANOVA results, ns (non-significant), ***3 (p < 0.0001), N = 30. (C) Immunoblots of co-immunoprecipitation experiments of CD31-GFP expressing cells incubated with 4 mg/mL CPB and lysed (1.5% digitonin). CD31-GFP was immunoprecipitated with anti-GFP beads, anti-myc beads were used as control. Immunoblots containing 5% input (In) and 50% eluate (elu) were probed with anti-GFP and anti-CPB antibodies. E-cadherin and VE-cadherin served as a control. See also Figures S3B–S3D.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Incubation, In Situ, Western Blot, Immunoprecipitation, Expressing, Control

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 4. CD31 Extracellular Ig6 Domain Is Required for CPB Targeting of Endothelial Cells (A) Top: schematic protein domain structures of CD31 with N-terminal signal peptide (SP), the extracellular domain (ED) containing the six Ig-like domains (Ig1– Ig6), the single transmembrane helix (TM), and the intracellular domain (ID). Below, corresponding exons. Constructs below show all different GFP fusion proteins used. Deleted exons are indicated as D in the protein name. Constructs that sensitized resistant cells are indicated in black, constructs that were non-sensitizing in gray. *Construct CD31D(3–7,10–16) did not localize at the plasma membrane and was not investigated further. (B and C) Viability of cell lines expressing indicated constructs after incubation with 1 mg/mL CPB (24 h, 37C) in % to untreated control cells. Data are represented as means (N = 8) ± SD. Two-way ANOVA, Sidak’s multiple comparison test, ***3 (p < 0.0001). (D) Co-immunoprecipitation experiment of bEndCD31ko cell lines expressing different CD31-GFP truncation mutants incubated with 4 mg/mL CPB for (20 min, 37C). IP: anti-GFP beads. Immunoblots containing 5% input (In) and 50% eluate (elu) and were probed with anti-GFP and CPB antibodies. The two upper blots show the same membrane probed with differently labeled fluorescent secondary antibodies. VE-cadherin served as a control on the same blot. See also Figures S4–S6.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Construct, Clinical Proteomics, Membrane, Expressing, Incubation, Control, Comparison, Immunoprecipitation, Western Blot, Labeling

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 5. Highly Conserved Region in Ig6 Is Required for CPB Cytotoxicity (A) Sequence alignments of the CD31 Ig6 domains of mus musculus, homo sapiens, and sus scrofa. Alignments were produced using EMBL-EBI server (https:// www.ebi.ac.uk/Tools/msa/clustalo). Identical residues are indicated by dark gray boxes, residues that belong to the same amino acid group by light gray boxes. Mutations are indicated with gray brackets if they were inconsequential or black if mutation diminished sensitivity to CPB. Black arrows indicate beta sheets, the dotted line indicates disulfide bond. (B) Viability of transduced HEK 293FT cells expressing five mouse CD31 Ig6 mutants (alanine and valine substitutions) after incubation with CPB (1 mg/mL, 24 h, at 37C) in % to untreated control cells. Data are represented as means (N = 12) ± SD. Two-way ANOVA, Sidak’s multiple comparison test, ***3 (p < 0.0001). (C) Schematic protein domain structures of species chimeras of mouse CD31 with either substituted pig or human Ig6 domain. (D) Viability of transduced HEK 293FT cells expressing indicated cimeras from C, mut indicates an alanine valine mutation at position 527–536 (mouse) or 537–546 (human and porcine). Data are represented as means (N = 16) ± SD. Two-way ANOVA, Sidak’s multiple comparison test, ***3 (p < 0.0001). See also Figure S6.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Sequencing, Produced, Mutagenesis, Expressing, Incubation, Control, Comparison

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 6. CPB Induces Rapid Content Leakage from CD31 Containing Liposomes (A) SDS PAGE (silver stained) and western blots showing purified mouse CD31 and CD31D8 (elu) from transduced HEK293T whole cell extracts (wcl). (B) Top, schematic illustrating the liposome floatation assay. Liposomes containing CD31 or CD31D8, or empty liposomes were incubated for 2 h with 10 mg/mL CPB and floated through a sucrose gradient by centrifugation. A sample before gradient centrifugation (crude) and the three fractions (top, middle, bottom) were analyzed by SDS-PAGE and western blotting. (C) Kinetic traces from carboxyfluorescein leakage assay of CD31-LUVs, CD31D8-LUVs, and empty LUVs treated with indicated dose of CPB or buffer as control (CD31, buffer)Arrowheads indicate addition of toxins or buffer after 20 s. Fluorescence was normalized to maximal fluorescence after complete solubilization of LUVs with detergent. (D) Kinetic traces from carboxyfluorescein leakage assay of CD31D8-LUVs and empty LUVs treated with indicated PFTs.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Liposomes, SDS Page, Staining, Western Blot, Incubation, Gradient Centrifugation, Control, Fluorescence