Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 1. Activation of ERK-1/2 by wild-type M3-muscarinic re- ceptors and the deletion mutant DLys370–Ser425. Stably trans- fected CHO cells expressing either the wild-type human M3-muscarinic receptor or the deletion mutant DLys370–Ser425 were stimulated for 5 min in the presence of varying concentrations of carbachol (CCH). The reaction was terminated by addition of lysis buffer, and ERK-1/2 activ- ity was determined. Shown are the concentration-response curves for wild-type receptor and two separate clones: clone 1 (A), clone 2 (B), expressing the DLys370–Ser425 receptor mutant. The data presented represent the mean 6 S.E. for three experiments.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Activation Assay, Mutagenesis, Stable Transfection, Expressing, Lysis, Concentration Assay, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 2. Serum-mediated ERK-1/2 responses. Stably transfected CHO cells expressing either the wild-type human M3-muscarinic recep- tor or the deletion mutant DLys370–Ser425 were stimulated for 20 min in the presence of varying concentrations of fetal calf serum. The reaction was terminated by addition of lysis buffer, and ERK-1/2 activity was determined. Shown are the concentration-response curves for wild-type receptor and two separate clones: clone 1 (A), clone 2 (B), expressing the DLys370–Ser425 receptor mutant. The data represent the mean 6 S.E. of at least three experiments.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Stable Transfection, Transfection, Expressing, Mutagenesis, Lysis, Activity Assay, Concentration Assay, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 5. Effect of the CK1a dominant negative mutant (F- CK1aK46R) and the 3i-loop peptide on the M3-muscarinic ERK- 1/2 response. CHO-m3 cells stably expressing recombinant M3-mus- carinic receptors (A) or native CHO-K1 cells (B), were transiently transfected with the CK1a dominant negative mutant, F-CK1aK46R (K46R) or the 3i-loop peptide (3i-loop) corresponding to Ser345–Leu463 of the third intracellular loop of the M3-muscarinic receptor or were sham transfected (Control). 48 h after transfection, cells were stimulated with 1 mM carbachol (CCH, A) or 1 mM phorbol 12,13-dibutyrate (PDBu, B), for 5 min. Reactions were terminated using lysis buffer, and ERK-1/2 activity was determined. The data represent the mean 6 S.E. of three experiments.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Dominant Negative Mutation, Stable Transfection, Expressing, Recombinant, Transfection, Control, Lysis, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 6. ERK-1/2 concentration-response curves in CHO-m3 cells transiently transfected with the CK1a dominant negative mutant (F-CK1aK46R) and the 3i-loop peptide. CHO-m3 cells sta- bly expressing recombinant M3-muscarinic receptors were transiently transfected with the CK1a dominant negative mutant, F-CK1aK46R (K46R) (A), or the 3i-loop peptide (3i-loop peptide, B) corresponding to Ser345–Leu463 of the third intracellular loop of the M3-muscarinic re- ceptor. 48 h after transfection cells were stimulated with varying con- centrations of carbachol (CCH) for 5 min. Reactions were terminated using lysis buffer, and ERK-1/2 activity was determined. The data represent the mean 6 S.E. of three experiments.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Concentration Assay, Transfection, Dominant Negative Mutation, Expressing, Recombinant, Lysis, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 7. Scheme of the mechanisms involved in the activation of the ERK-1/2 pathway by M3-muscarinic receptors. Our data have identified two mechanisms involved in the activation of the ERK-1/2 pathway by M3-muscarinic receptors expressed in CHO cells. Mecha- nism 1 is PKC-dependent and is essential in the activation of ERK-1/2. Inhibition of Mechanism 1 (e.g. inhibition of PKC with Ro-318220) prevents activation of ERK-1/2 despite the fact that Mechanism 2 is still intact. Mechanism 2, therefore, will not elicit an ERK-1/2 response alone. However, Mechanism 2 does operate in concert with Mechanism 1 to give a full ERK-1/2 response. Hence a receptor that is only able to activate Mechanism 1 (i.e. the DLys370–Ser425 receptor mutant or the wild-type M3-muscarinic receptor expressed together with the 3i-loop peptide or F-CK1aK46R) will give a less than maximal ERK-1/2 re- sponse. CK1a, casein kinase 1a; DAG, diacylglycerol; InsP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Activation Assay, Inhibition, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mesenchymal Stromal Cell-Conditioned Medium for Skin Diseases: A Systematic Review

doi: 10.3389/fcell.2021.654210

Figure Lengend Snippet: Studies regarding mesenchymal stromal cell-conditioned medium for treating wounds in animal models.

Article Snippet: , Human amnion , Amniocentesis , Flow cytometry (CD73, CD45, CD31, CD105, CD44, CD34, CD90, CD29, SSEA-3, SSEA-4, EP-CAM, HLA-DR). Chondrogenic, osteogenic, and adipogenic differentiation , MSCs of passage 2 at 90% confluence were used. CM was collected , Murine. Full-thickness excisional skin wounds, 10 mm, on the dorsum , 50 μl subcutaneous injected into the surrounding tissues of the wound bed at four sites - PBS (control) - MSC-CM - Murine LOXL2 (5 μg, Sino Biologicals, China) ( n = 4/group) , 12 , Macroscopic appearance (photography), histology (HE, TC) , Wound sizes were significantly reduced in the LOXL2 and MSC-CM groups compared with controls ( p < 0.05) , MSC-CM and LOXL2 enhanced wound healing. Epidermis of the MSC-CM and LOXL2 group mice resembled normal skin and the keratinocytes were well organized and tightly arranged. These treatments also significantly reduced fibrosis and improved keratinocyte proliferation. No adverse events were reported.

Techniques: Flow Cytometry, Expressing, Isolation, Injection, Transfection, Recombinant, Construct, Dissection, Transduction, Plasmid Preparation, Fluorescence, Marker, Cell Culture, Staining, Activation Assay, Migration, Enzyme-linked Immunosorbent Assay, Functional Assay, Generated, Immunofluorescence, Immunohistochemistry-IF, Immunoprecipitation

Journal: Oncotarget

Article Title: Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc-γ receptor engagement

doi: 10.18632/oncotarget.19090

Figure Lengend Snippet: A . Biotinylated farletuzumab and its (Fab’) 2 fragment, but not its Fc fragment bind immobilized sCA125. 96-well plates were coated with 15 KU/mL sCA125 or human serum albumin (HSA) and probed with biotin-labeled farletuzumab, (Fab’) 2 or Fc fragments. B . Farletuzumab is able to compete for farletuzumab-biotin binding to sCA125. C . sCA125 suppresses Fc receptor binding to farletuzumab. Farletuzumab was incubated alone or with sCA125 or HSA and probed with biotinylated-CD16a, -CD32a or -CD64a Fc receptor and graphed as a percent inhibition of Fc receptor binding compared to control. sCA125 caused a significant decrease of CD16a binding to farletuzumab as compared to controls (46%, p < 0.0002). Reduction in farletuzumab binding to CD32a Fc receptors was also significant (12%, p < 0.003) while minimal inhibition was observed by sCA125 on farletuzumab binding to CD64a Fc receptor (1%, p = 0.772). Similar reactions were probed with anti-human IgG-HRP to confirm incubation of farletuzumab with sCA125 did not result in less farletuzumab binding to wells . D . BRA assay using CHO cells expressing human Fc activating receptors show only farletuzumab-CD16a interactions are disrupted by sCA125. E . Farletuzumab has a significant increase in binding to the mCA125 producing OVCAR parental as compared to mCA125-suppressed isogenic OVCAR-KD1 or mCA125-null CHO membranes. F . sCA125 can bind to subsets of other humanized and fully human mAbs. Seven purified mAbs were biotinylated and used to probe wells coated with 15 KU/mL sCA125 or HSA. While all antibodies were able to robuslty and equally bind their respective target antigen, only a subset were able to specifically bind sCA125 as compared to HSA controls. All ELISAs were done in at least triplicate and different lots of sCA125 or cell membrane preparations were used with similar results. In panels A and B *** p < 0.0001; **** p < 0.00007; ***** p < 0.000001, panels E and F *** p < 0.0001; **** p < 0.00001.

Article Snippet: Human CD16a-, CD32a- and CD64a-biotin probes were purchased from Sino Biological Inc. Biotinylated CA125 or HSA were generated as previously described [ ].

Techniques: Labeling, Binding Assay, Incubation, Inhibition, Expressing, Purification

Journal: The Journal of Allergy and Clinical Immunology

Article Title: ARGX-117, a therapeutic complement inhibiting antibody targeting C2

doi: 10.1016/j.jaci.2020.08.028

Figure Lengend Snippet: ARGX-117 does not interact with C1q (A) , CD16 (B) , or CD32a (C) , and binds stronger to FcRn than to wild-type (WT) IgG 1 at pH 6.0 (D). A, ELISA plates were coated with ARGX-117, incubated with serum, and C1q was detected with anti-C1q. B-D, Plates were coated with NeutrAvidin and incubated with biotinylated CD16 (B) , CD32a (C) , or FcRn (D) . Subsequently, plates were incubated with ARGX-117. Bound ARGX-117 was then detected with poly-HRP-labeled goat anti-human IgG antibody.

Article Snippet: Plates were then incubated for 1 hour with anti-human CD32a (His Tagged; Sino Biological) and incubated with serial dilutions of ARGX-117 or ARGX-18E2 for 1 hour.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Labeling