Journal: STAR protocols

Article Title: Enzyme-free isolation of mesenchymal stem cells from decidua basalis of the human placenta.

doi: 10.1016/j.xpro.2023.102498

Figure Lengend Snippet: Figure 2. Immunophenotypic characterization of isolated PL-MSCs The antibodies used were (A) human CD73, (B) human CD90 (C) human CD105 (D) human CD166 (E) human CD34 (F) human HLA-DR and their respective isotype controls. Open histogram indicates background signal, while, shaded histogram represents positive reactivity with the indicated antibodies. Table shows percentage of cells expressing the respective surface markers (n = 3) in mean G SEM.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies PE Mouse Anti-Human CD73 (1:33) BD Pharmingen Catalog No 550257 PE Mouse Anti-Human CD90 (1:33) BD Pharmingen Catalog No 555596 CD105 PE-conjugated Antibody (1:50) R&D Systems Catalog No FAB10971P PE Mouse Anti-Human CD34 (1:50) BD Pharmingen Catalog No 550761 PE Mouse Anti-Human CD166 (1:50) BD Pharmingen Catalog No 559263 FITC Mouse Anti-Human HLA-DR (1:33) BD Pharmingen Catalog No 555811 PE Mouse IgG, k Isotype Control (1:33 as isotype for CD90, 1:800 as isotype for CD34/ CD73/CD166) BD Pharmingen Catalog No 550617 Mouse IgG1 PE-conjugated Antibody (1:50) R&D Systems Catalog No IC002P FITC Mouse IgG2a, k Isotype Control (1:33) BD Pharmingen Catalog No 555573 Vimentin (D21H3) XP Rabbit mAb Antibody (1:150) Cell Signaling Technology Catalog No 5741S Human STRO-1 antibody (1:100) R&D Systems Catalog No MAB1038 Oct-4 (D705Z) Mouse mAb (1:400) Cell Signaling Technology Catalog No 75463S Alexa Fluor 488 goat anti-mouse (1:500) Invitrogen Catalog No A11001 Alexa Fluor 568 goat anti-rabbit (1:500) Invitrogen Catalog No A11011 Alexa Fluor 488 anti-rabbit (1:500) Cell Signaling Technology Catalog No 4412S Biological samples Human placenta N/A N/A Chemicals, peptides, and recombinant proteins 0.9% w/v Saline Kunal Remedies Pvt.

Techniques: Isolation, Expressing

Journal: Cells

Article Title: Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

doi: 10.3390/cells8091082

Figure Lengend Snippet: Protein–protein association between galectin-3 and endoglin. ( A – C ). Co-immunoprecipitation of galectin-3 and endoglin. CHO-K1 cells were transiently transfected with pcEXV-Ø (Ø), pcEXV–HA–EngFL (Eng) and pcDNA3.1–Gal-3 (Gal3) expression vectors. ( A ) Total cell lysates (TCL) were analyzed by SDS-PAGE under reducing conditions, followed by Western blot (WB) analysis using specific antibodies to endoglin, galectin-3 and β-actin (loading control). Cell lysates were subjected to immunoprecipitation (IP) with anti-endoglin ( B ) or anti-galectin-3 ( C ) antibodies, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin or anti-galectin-3 antibodies, as indicated. Negative controls with an IgG2b ( B ) and IgG1 ( C ) were included. ( D ) Protein-protein interactions between galectin-3 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 7.3 µM recombinant human galectin-3/6xHis at the C-terminus (LGALS3), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.

Article Snippet: Two replicate analyses were hybridized with purified recombinant human soluble endoglin (sEng; Glu26-Gly586; 1097-EN, R&D Systems).

Techniques: Immunoprecipitation, Transfection, Expressing, SDS Page, Western Blot, Control, Protein-Protein interactions, Recombinant, Protein Binding, Negative Control

Journal: Cells

Article Title: Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

doi: 10.3390/cells8091082

Figure Lengend Snippet: Protein–protein association between TRIM21 and endoglin. ( A – E ) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin ( A ). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin ( B ). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL ( E ) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin ( C ). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included ( D ). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. ( F ) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.

Article Snippet: Two replicate analyses were hybridized with purified recombinant human soluble endoglin (sEng; Glu26-Gly586; 1097-EN, R&D Systems).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Negative Control, Transfection, Expressing, Protein-Protein interactions, Recombinant, Protein Binding