Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 interferes with complement regulation within the glomerulus

doi: 10.1016/j.jbc.2025.110657

Figure Lengend Snippet: Interactions between annexin A2 and the different glomerular cell types. A , expression of annexin A2 on mesangial cells, glomerular endothelial cells (GEnC), and podocytes was examined by flow cytometry. Annexin A2 was detected on all three cell types. B , immunofluorescence microscopy for annexin A2 in GEnCs indicated that the protein is also expressed within the cells. C , When injured/apoptotic and healthy cells were distinguished by side scatter and forward scatter, more annexin A2 was seen on the surface of the apoptotic mesangial cells and podocytes, whereas expression was comparable on the injured and healthy GEnC populations. D , when rA2 was added to glomerular cells exposed to 10% normal mouse serum, a trend towards more C3b was deposited on the surface of GEnCs compared to cells exposed to serum without rA2, and greater C3 was deposited on podocytes. Groups were compared using an unpaired Student t test with a two-tailed p value.

Article Snippet: Antibodies used in the experiments included a rabbit polyclonal antibody to human annexin A2 (Proteintech; #11256-1-AP, 1:250 dilution), IgG1 monoclonal anti-AnxA2 clone Z014 (Invitrogen; #034400, 2 μg per sample), IgG1 isotype control clone MG1-45 (Abcam; # ab18448, 2 μg per sample), a Cy3-conjugated polyclonal goat anti-mouse IgG (Jackson Immuno; 1:100 dilution), an Alexa Fluor Plus 647 labeled goat anti-mouse IgG (Invitrogen; #A32728, 1:1000 dilution), a FITC-labeled goat polyclonal antibody to mouse C3 (MP Biomedicals; #55500; 10 μg/ml for tissue staining), Digoxin (DIG) labeled anti-mouse C9 (generously provided by Cees van Kooten, Leiden University Medical Center; 2 μg/ml for tissue staining), DyLight 405 IgG fraction monoclonal mouse anti-Digoxin (Jackson Immuno; #200-472-156, 2.7 μg/ml for tissue staining), and MECA32, a pan-endothelial cell antigen antibody (Novus Biologicals; NB100-77668, 0.25 μg/100 μl reaction containing 10 6 cells).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 interferes with complement regulation within the glomerulus

doi: 10.1016/j.jbc.2025.110657

Figure Lengend Snippet: Recombinant annexin A2 increases glomerular C3b deposition in vivo . A , fH +/− mice were injected with 75 μg recombinant annexin A2. After 24 h, the kidneys were harvested and examined by immunofluorescence microscopy. Original magnification ×600. B , deposition of C3b and iC3b/C3d was quantified, and the abundance of both activation fragments was increased compared to mice injected with phosphate-buffered saline (PBS). Injection of the mice with rA2 did not increase C9 deposits in the glomeruli. C , kidneys of rA2-injected and control mice were digested with collagenase. Endothelial cells were identified by surface expression of MECA32, and C3b deposits on the cells were measured by flow cytometry. D , endothelial cells from rA2-injected mice had significantly more C3b deposited on their surface compared to PBS-injected mice. Groups were compared using an unpaired Student t test with a two-tailed p value.

Article Snippet: Antibodies used in the experiments included a rabbit polyclonal antibody to human annexin A2 (Proteintech; #11256-1-AP, 1:250 dilution), IgG1 monoclonal anti-AnxA2 clone Z014 (Invitrogen; #034400, 2 μg per sample), IgG1 isotype control clone MG1-45 (Abcam; # ab18448, 2 μg per sample), a Cy3-conjugated polyclonal goat anti-mouse IgG (Jackson Immuno; 1:100 dilution), an Alexa Fluor Plus 647 labeled goat anti-mouse IgG (Invitrogen; #A32728, 1:1000 dilution), a FITC-labeled goat polyclonal antibody to mouse C3 (MP Biomedicals; #55500; 10 μg/ml for tissue staining), Digoxin (DIG) labeled anti-mouse C9 (generously provided by Cees van Kooten, Leiden University Medical Center; 2 μg/ml for tissue staining), DyLight 405 IgG fraction monoclonal mouse anti-Digoxin (Jackson Immuno; #200-472-156, 2.7 μg/ml for tissue staining), and MECA32, a pan-endothelial cell antigen antibody (Novus Biologicals; NB100-77668, 0.25 μg/100 μl reaction containing 10 6 cells).

Techniques: Recombinant, In Vivo, Injection, Immunofluorescence, Microscopy, Activation Assay, Saline, Control, Expressing, Flow Cytometry, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 interferes with complement regulation within the glomerulus

doi: 10.1016/j.jbc.2025.110657

Figure Lengend Snippet: Mice deficient in annexin A2 are protected from cyclosporine-induced kidney injury. A , mice with targeted deletion of the gene for annexin A2 ( A2 −/− mice) and wild-type control mice were injected subcutaneously with cyclosporine (CsA) for 2 weeks. A , serum urea nitrogen (SUN) was measured as a marker of kidney function. SUN levels were significantly increased in wild-type mice injected with CsA, but did not increase in the A2 −/− mice. Glomerular deposition of C3b ( B ) and iC3b/C3d ( C ) were quantified by fluorescence microscopy, and the abundance of both activation fragments were similar in the wild-type and A2 −/− mice. D , representative images stained for C3b ( green ) and iC3b/C3d ( red ) are shown. Original magnification ×200. Glomeruli are indicated with arrowheads . E , endothelial extracellular vesicles (EVs) were isolated from the plasma of CsA treated mice, and C3b deposits on the surface of the EVs were measured by flow cytometry. The abundance of C3b on the EVs from wild-type mice were increased in mice treated with CsA, whereas it did not increase in A2 −/− mice treated with CsA. F , the number of endothelial EVs per μl of plasma was significantly higher in the A2 −/− mice treated with CsA compared to the other groups ( p < 0.01 for A2 −/− CsA versus the other groups). Groups were compared using an ANOVA, with a Tukey’s multiple comparison test.

Article Snippet: Antibodies used in the experiments included a rabbit polyclonal antibody to human annexin A2 (Proteintech; #11256-1-AP, 1:250 dilution), IgG1 monoclonal anti-AnxA2 clone Z014 (Invitrogen; #034400, 2 μg per sample), IgG1 isotype control clone MG1-45 (Abcam; # ab18448, 2 μg per sample), a Cy3-conjugated polyclonal goat anti-mouse IgG (Jackson Immuno; 1:100 dilution), an Alexa Fluor Plus 647 labeled goat anti-mouse IgG (Invitrogen; #A32728, 1:1000 dilution), a FITC-labeled goat polyclonal antibody to mouse C3 (MP Biomedicals; #55500; 10 μg/ml for tissue staining), Digoxin (DIG) labeled anti-mouse C9 (generously provided by Cees van Kooten, Leiden University Medical Center; 2 μg/ml for tissue staining), DyLight 405 IgG fraction monoclonal mouse anti-Digoxin (Jackson Immuno; #200-472-156, 2.7 μg/ml for tissue staining), and MECA32, a pan-endothelial cell antigen antibody (Novus Biologicals; NB100-77668, 0.25 μg/100 μl reaction containing 10 6 cells).

Techniques: Control, Injection, Marker, Fluorescence, Microscopy, Activation Assay, Staining, Isolation, Clinical Proteomics, Flow Cytometry, Comparison