Mouse MFG-E8 Antibody

Detection of Mouse MFG-E8 by Western Blot.

Western blot shows lysates of mouse mammary gland tissue. PVDF membrane was probed with 0.25 µg/mL of Goat Anti-Mouse MFG-E8 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2805) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). A specific band was detected for MFG-E8 at approximately 60-70 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Mouse MFG‑E8 by Simple Western^TM.

Simple Western lane view shows lysates of recombinant mouse (rm) MFG-E8 , loaded at 50 ng/mL. A specific band was detected for MFG-E8 at approximately 87 kDa (as indicated) using 50 µg/mL of Goat Anti-Mouse MFG-E8 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2805) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Human MFG-E8 by Western Blot

MFGE8 and NO Are Produced and Secreted by Co-cultured BV2 Microglia Only When Co-cultured in Contact with pFTAA+Neurons(A and B) Representative blot (A) ofMFGE8 in BV2 cells lysates cultured alone or co-cultured in direct contact, or via atranswell, with DRG neurons from 5-month-old P301S-tau, 5-month-old C57, 5-month-old Alz17 mice, or 2-month-old P301S mice.Asterisk: either another isoform ofMFGE8 or a breakdown product. (B) Densitometry of MFGE8 expression normalized to beta-actin; significantly more MFGE8 is produced only in contact co-cultures containing pFTAA+ neurons. Mean± SD, n = 3 independent experiments (*p < 0.05), twoway ANOVA, Dunnett’s correction.(C and D) Elevated MFGE8 (C) or NO (D) in medium conditioned by BV2 cells co-cultured in contact with DRG neurons from5-month-old P301S mice compared with 5-month-old Alz17, 5-month- old C57, or 2-month-old P301S mice (MFGE8: ****p < 0.0001versus all; NO: ****p < 0.0001 versus 5-month-old C57 or 2-month-old P301S mice; ***p < 0.001 versus 5-month-oldAlz17 mice). N.D., none detected. Mean ± SD, n = 3 independent experiments, two-way ANOVA, Bonferroni corrected.(E and F) Elevated MFGE8 (E) or NO (F) in medium conditioned by primary microglia from C57 mice co-cultured in contactwith DRG neurons from 5-month-old P301S mice; microglia from MFGE8 KO mice are negative controls. Mean ± SD, n = 3independent microglial preparations, one-way ANOVA, **p < 0.01 (MFGE8), *p < 0.05 (NO).(G) Increased MFGE8 immunostaining intensity in frontal motor cortex of 5-month-old P301S mice compared with 5-month-oldC57 mice; MFGE8 KO mouse is negative control; 25 μm section at inter- aural 5.12 mm, bregma 1.32 mm; brown, DAB; blue,cresyl violet. Scale bar, 130 mm. Inset, 65 μm.(H and I) Elevated MFGE8 (H) in 5-month-old P301S-tau brains. Lysatesfrom cortex(Ctx), brain stem (BS), and cerebellum(Cb) of 5-month-old C57, 5-month-old P301S-tau, and 5-month-old Alz17 mice probed with anti-MFGE8; MFGE8 KO brain lysate isnegative control. rec, recombinant mouse MFGE8. (I) Densitometry of MFGE8 expression normalized to beta-actin. Significantlyhigher MFGE8 expression in P301S versus C57BU6 Ctx (**p < 0.01), BS (****p < 0.0001), Cb (**p < 0.01), andP301SversusALz17BS (****p < 0.0001). Mean ± SD, n = 3 independent preparations, two-way ANOVA, Bonferronicorrected.(J) Elevated MFGE8 expression in brain extracts from cortex (Ctx) of FTDP-17T patients with two differentMAPT mutations (P301L, +3) or sporadic Pick’s disease but not in extracts from patients with C9orf72hexanucleotide expansions and TDP-43 aggregates. No expression is found in the cerebellum, where tau pathology is absent in allcases. Human milk MFGE8 is a positive control. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/30134156), licensed under a CC-BY license. Not internally tested by R&D Systems.