mouse cd47  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: CD47-blocking antibody confers metabolic benefits against obesity

doi: 10.1016/j.xcrm.2025.102089

Figure Lengend Snippet: Administration of CD47-blocking antibody confers metabolic protective effects in mice under HFD condition (A) Correlation of CD47 expression with BMI in individuals with type 2 diabetes (T2D). (B) Correlation between CD47 expression and HOMA-IR in obese individuals. (C and D) CD47 mRNA and protein expression levels in skeletal muscle of control and obese mice ( n = 4). Expression levels quantified, GAPDH as control. (E and F) CD47 mRNA and protein expression in sedentary and exercise-trained mice after 8 weeks ( n = 4). Expression quantified, GAPDH as control. (G) Schematic of antibody injection protocol: CD47-blocking antibody (CD47 Ab) or control IgG antibody (Cont) every two days under HFD. (H) Bodyweight changes in mice injected with CD47 Ab or Cont on HFD ( n = 6 for Cont and n = 7 for CD47 Ab). (I and J) Body composition measured by NMR in mice ( n = 6 for Cont and n = 8 for CD47 Ab). (K) Intraperitoneal glucose tolerance test (IPGTT) and area under the curve (AUC) after 5 weeks HFD administration ( n = 6). (L–N) Oxygen consumption, carbon dioxide emissions, and energy expenditure measured using metabolic cage ( n = 5 for Cont and n = 6 for CD47 Ab). (O and P) Spontaneous food intake and physical activity ( n = 5 for Cont and n = 6 for CD47 Ab). (Q and R) Running distance and duration measured on a treadmill ( n = 6). Data are presented as means ± SEM and analyzed by two-tailed Student’s t test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant).

Article Snippet: Milk (5%) in TBST was used to block members for 1 h at room temperature, then the members were washed with TBST three times (5 min each time) and incubated with primary antibodies against P-AMPK (Thr172, 2535), AMPK (2532), P-HSP90α (Thr5/7, 3488), P-ACC (3661), ACC (3676), p-DNA-PK (Ser2056, 68716), COXIV (4850), cyto C (4280), Acetylated-Lysine (9814) (Cell Signaling Technology Inc.), SIRT1 (04-1557, Millipore), PGC-1α (ab54481, Abcam), HSP90α (A5006, ABclonal), DNA-PK (ab70250, Abcam), AMPKγ3 (A14132, ABclonal), AMPKγ1 (A22024, ABclonal), CD47 (AF1866-SP, R&D systems), and GAPDH (AB0037, Abways) in 5% BSA at 4°C overnight.

Techniques: Blocking Assay, Expressing, Control, Injection, Activity Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: CD47-blocking antibody confers metabolic benefits against obesity

doi: 10.1016/j.xcrm.2025.102089

Figure Lengend Snippet: CD47-blocking antibody promotes skeletal muscle AMPK activation and improves mitochondrial function (A) AMPK phosphorylation in skeletal muscle, WAT, BAT, liver, hypothalamus (Hypo), and kidney ( n = 6). (B) ACC phosphorylation in skeletal muscle after antibody injection. GAPDH as control. (C) PGC-1α protein levels in skeletal muscle post-injection. GAPDH as control. (D) Western blot analysis of AMPK, ACC phosphorylation, and PGC-1α in skeletal muscle of mice injected with CD47 Ab (20 mg/kg), Cont (20 mg/kg), and AMPK inhibitor (Compound C, 10 mg/kg) ( n = 3). (E and F) AMPK phosphorylation and cytochrome c (cyto c) protein expression in myotubes after antibody treatment. GAPDH as control. (G) Seahorse mitochondrial stress test in myotubes treated with CD47 Ab. (H–K) AMPK, ACC phosphorylation, and PGC-1α expression in myotubes with Cd47 knockdown or overexpression. (L) Seahorse mitochondrial stress test in myotubes with Cd47 knockdown. Data are presented as means ± SEM and analyzed by two-tailed Student’s t test (A and G) and two-way repeated measures (RM) ANOVA (D and L). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Milk (5%) in TBST was used to block members for 1 h at room temperature, then the members were washed with TBST three times (5 min each time) and incubated with primary antibodies against P-AMPK (Thr172, 2535), AMPK (2532), P-HSP90α (Thr5/7, 3488), P-ACC (3661), ACC (3676), p-DNA-PK (Ser2056, 68716), COXIV (4850), cyto C (4280), Acetylated-Lysine (9814) (Cell Signaling Technology Inc.), SIRT1 (04-1557, Millipore), PGC-1α (ab54481, Abcam), HSP90α (A5006, ABclonal), DNA-PK (ab70250, Abcam), AMPKγ3 (A14132, ABclonal), AMPKγ1 (A22024, ABclonal), CD47 (AF1866-SP, R&D systems), and GAPDH (AB0037, Abways) in 5% BSA at 4°C overnight.

Techniques: Blocking Assay, Activation Assay, Phospho-proteomics, Injection, Control, Western Blot, Expressing, Knockdown, Over Expression, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: CD47-blocking antibody confers metabolic benefits against obesity

doi: 10.1016/j.xcrm.2025.102089

Figure Lengend Snippet: CD47 LOF in skeletal muscle promotes AMPK activation (A) Schematic of CD47-knockout (CD47 −/− ) mouse model. (B) Growth curves of WT and CD47 −/− mice ( n = 6). (C) IPGTT and AUC in WT and CD47 −/− mice ( n = 6). (D and E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes in skeletal muscle of CD47 −/− and exercise mice. (F) AMPK and ACC phosphorylation in skeletal muscle of WT and CD47 −/− mice. (G) PGC-1α, cyto c, and oxidase (COXIV) expression levels in skeletal muscle of WT and CD47 −/− mice. (H and I) ATPase and CS activities in skeletal muscle of WT and CD47 −/− mice ( n = 7). (J) Schematic of MCK-CD47 mouse model. (K) Growth curves of MCK-CD47 mice on HFD ( n = 10 for Con and n = 6 for MCK-CD47). (L) IPGTT and AUC in MCK-CD47 mice after 3 months of HFD ( n = 10 for Con and n = 6 for MCK-CD47). (M) AMPK phosphorylation and PGC-1α expression in skeletal muscle of MCK-CD47 mice. (N) CS activity in skeletal muscle of MCK-CD47 mice ( n = 11 for Con and n = 7 for MCK-CD47). (O) Schematic of HSA-CD47 mouse model. (P and Q) Growth curves and IPGTT of HSA-CD47 mice on HFD ( n = 8 for Con and n = 6 for HSA-CD47). (R and S) Running distance and duration of HSA-CD47 mice on motor treadmill ( n = 9 for Con and n = 7 for HSA-CD47). (T) CS activity in skeletal muscle of HSA-CD47 mice ( n = 9 for Con and n = 7 for HSA-CD47). (U) AMPK phosphorylation and PGC-1α expression in skeletal muscle of HSA-CD47 mice. (V) Western blot analysis of CD47 and AMPK phosphorylation in skeletal muscle of mice injected with AAV expressing GFP or CD47. Data are presented as means ± SEM and analyzed by two-tailed Student’s t test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant).

Article Snippet: Milk (5%) in TBST was used to block members for 1 h at room temperature, then the members were washed with TBST three times (5 min each time) and incubated with primary antibodies against P-AMPK (Thr172, 2535), AMPK (2532), P-HSP90α (Thr5/7, 3488), P-ACC (3661), ACC (3676), p-DNA-PK (Ser2056, 68716), COXIV (4850), cyto C (4280), Acetylated-Lysine (9814) (Cell Signaling Technology Inc.), SIRT1 (04-1557, Millipore), PGC-1α (ab54481, Abcam), HSP90α (A5006, ABclonal), DNA-PK (ab70250, Abcam), AMPKγ3 (A14132, ABclonal), AMPKγ1 (A22024, ABclonal), CD47 (AF1866-SP, R&D systems), and GAPDH (AB0037, Abways) in 5% BSA at 4°C overnight.

Techniques: Activation Assay, Knock-Out, Phospho-proteomics, Expressing, Activity Assay, Western Blot, Injection, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: CD47-blocking antibody confers metabolic benefits against obesity

doi: 10.1016/j.xcrm.2025.102089

Figure Lengend Snippet: CD47-blocking antibody promotes AMPK activation by reducing phosphorylation of HSP90α (A) Strategy for analyzing CD47-interacting proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). (B) Endogenous interactions between CD47 and HSP90α detected in myotubes by western blotting. (C and D) HSP90α and DNA-PK phosphorylation in Cd47 knockdown or overexpressing myotubes. (E) DNA-PK inhibitor treatment (10 μM) effects on HSP90α and DNA-PK phosphorylation in Cd47 -overexpressing myotubes. (F) HSP90α and DNA-PK phosphorylation in myotubes treated with CD47-blocking antibody. (G) HSP90α phosphorylation and AMPK activation after treatment with tanespimycin (17-AAG). (H) AMPK phosphorylation in Cd47 -overexpressing myotubes treated with 17-AAG. (I) AMPK phosphorylation after lentiviral treatment with mutated HSP90α phosphorylation sites. (J) The inhibitory effect of Cd47 overexpression on AMPK activation was blocked by mutated HSP90α phosphorylation sites. (K) Schematic: CD47-blocking antibody reduces HSP90α phosphorylation, promoting AMPK activation. Data are presented as means ± SEM and analyzed by two-way RM ANOVA (C, right) and Student’s t test (D, right). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Milk (5%) in TBST was used to block members for 1 h at room temperature, then the members were washed with TBST three times (5 min each time) and incubated with primary antibodies against P-AMPK (Thr172, 2535), AMPK (2532), P-HSP90α (Thr5/7, 3488), P-ACC (3661), ACC (3676), p-DNA-PK (Ser2056, 68716), COXIV (4850), cyto C (4280), Acetylated-Lysine (9814) (Cell Signaling Technology Inc.), SIRT1 (04-1557, Millipore), PGC-1α (ab54481, Abcam), HSP90α (A5006, ABclonal), DNA-PK (ab70250, Abcam), AMPKγ3 (A14132, ABclonal), AMPKγ1 (A22024, ABclonal), CD47 (AF1866-SP, R&D systems), and GAPDH (AB0037, Abways) in 5% BSA at 4°C overnight.

Techniques: Blocking Assay, Activation Assay, Phospho-proteomics, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Knockdown, Over Expression