Journal: Antioxidants

Article Title: Inhibiting NLRP3 Inflammasome Activation by CY-09 Helps to Restore Cerebral Glucose Metabolism in 3×Tg-AD Mice

doi: 10.3390/antiox12030722

Figure Lengend Snippet: CY-09 reversed the expression and distribution of Aβ related proteins in 3×Tg-AD mice. ( a – h ) Western blot analysis of amyloid precursor protein (APP), beta-site app cleaving enzyme 1 (BACE1), sAPPα, sAPPβ, Aβ1-42, post-synaptic density protein 95 (PSD95), and synaptophysin in NTg, NTg + CY-09, 3×Tg-AD, and 3×Tg-AD + CY-09 mice. (n = 6, mean ± SD, one-way ANOVA and Bonferroni post hoc test; * p < 0.05, ** p < 0.01 vs. NTg mice, & p < 0.05, && p < 0.01 vs. NTg + CY-09 mice, # p < 0.05, ## p < 0.01 vs. 3×Tg-AD mice). ( i ) Distribution of Aβ in the CA3 region and cortex of the four groups of mice (scale bar: 100 μm and 250 μm; arrows indicate Aβ in 3×Tg-AD mice).

Article Snippet: The antibodies against AS160 (Cat#ab189890), p-AS160-T642 (Cat#ab131214), HK1 (Cat#ab150423), PDHE1α (Cat#ab168379), COX IV (Cat#ab16056), APP (Cat#ab32136), BACE1 (Cat#ab108394), PSD95 (Cat#ab18258), synaptophysin (Cat#ab32127), tau5 (Cat#ab80579), p-tau-Ser404 (Cat#ab92676), MDA (Cat#ab27642), GPX4 (Cat#ab125066), and HMGCS1 (Cat#ab155787) were purchased from Abcam plc., Cambridge, UK.

Techniques: Expressing, Western Blot

Journal: The Journal of endocrinology

Article Title: Amyloid Precursor Protein in Pancreatic Islets

doi: 10.1530/JOE-17-0122

Figure Lengend Snippet: (A) Whole pancreas was collected from C57BL/6 (WT), APP−/−, and APP/PS1 mice (n=7) and lysed in RIPA buffer. 20 μg of protein was resolved by 10% SDS-PAGE and western blotted using anti-APP (Y188) antibody or anti-GAPDH antibody as a loading control. Normalized optical density was averaged +/− SD, *p<0.05. (B) Pancreas was harvested from C57BL/6 (WT), APP/PS1, and APP−/− at 2 months (n=7) and 12 months (n=4) of age. Tissue was fixed in 4% paraformaldehyde and cryosectioned into 10 μm serial sections. Immunohistochemistry was performed using anti-APP (Y188) antibody. Vector VIP was used as the chromogen. (C) Immunohistochemistry was performed on human pancreatic tissue. Pancreas from a healthy 77 year old male donor, 68 year old male type 2 diabetic donor and a 66 year old male with pancreatic adenocarcinoma was probed using anti-APP antibody (Y188) with vector VIP used as the chromogen. (D) Representative western blot of protein lysates from cultured pancreatic islets from WT, APP−/−, and APP/PS1 mice. Islet protein was resolved by 10% SDS-PAGE, transferred to PVDF, and immunoblotted using anti-APP antibody (Y188). Islets from a healthy 55 year old male donor were cultured then lysed in RIPA buffer and resolved using 10% SDS-PAGE then transferred to PVDF membrane and western blotted using anti-APP antibody, 22C11.

Article Snippet: Antibodies against full length APP (ab32136), BACE2 (ab8025), glucagon (ab10988), IDE (ab32216) and the insulin receptor (ab69508) were purchased from Abcam (Cambridge, UK).

Techniques: SDS Page, Western Blot, Control, Immunohistochemistry, Plasmid Preparation, Cell Culture, Membrane

Journal: The Journal of endocrinology

Article Title: Amyloid Precursor Protein in Pancreatic Islets

doi: 10.1530/JOE-17-0122

Figure Lengend Snippet: Immunofluorescent staining was performed using (A) APP/PS1 transgenic mouse and (B) human tissue cryosections (10μm). Anti-APP (Y188) and insulin primary antibodies with Alexa Flour 488 and Alexa Flour 594 fluorophore conjugated secondary antibodies were used for visualization with DAPI counterstain.

Article Snippet: Antibodies against full length APP (ab32136), BACE2 (ab8025), glucagon (ab10988), IDE (ab32216) and the insulin receptor (ab69508) were purchased from Abcam (Cambridge, UK).

Techniques: Staining, Transgenic Assay

Journal: The Journal of endocrinology

Article Title: Amyloid Precursor Protein in Pancreatic Islets

doi: 10.1530/JOE-17-0122

Figure Lengend Snippet: (A) Mouse pancreas and hippocampus tissue was lysed in RIPA buffer (n=4/strain), while islets (n=6/strain) were pelleted and lysed in Triton X-100 buffer to yield detergent soluble Aβ extract. Extract was used to quantify both human Aβ 1-40 and human Aβ 1-42 by ELISA. APP−/− animals served as a negative control to measure the background of the assay. Data is expressed as mean values +/−SD, *p<0.05 from WT. (B) Immunohistochemistry was performed on 10μm cryosections of WT, APP−/−, and APP/PS1 murine pancreas as well as human healthy control, type 2 diabetes (T2D), and adenocarcinoma pancreas tissue. Anti-Aβ antibody (4G8) was used with Vector VIP as the chromogen.

Article Snippet: Antibodies against full length APP (ab32136), BACE2 (ab8025), glucagon (ab10988), IDE (ab32216) and the insulin receptor (ab69508) were purchased from Abcam (Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Immunohistochemistry, Control, Plasmid Preparation

Journal: The Journal of endocrinology

Article Title: Amyloid Precursor Protein in Pancreatic Islets

doi: 10.1530/JOE-17-0122

Figure Lengend Snippet: 2 month old WT, APP−/−, and APP/PS1 mice were subjected to glucose tolerance testing. Prior to testing, mice were fasted four hours and then received 2g/kg glucose by oral gavage. (A) Blood glucose was measured at intervals by blood glucose meter (n=4-7). (B) The area under the curve (AUC) from glucose tolerance testing of each mouse was measured and the results from each strain are graphed. Each strain was normalized to the mean AUC of the WT strain. Whole mouse pancreas was lysed using RIPA buffer. (C) Insulin (n=6-7) and (D) glucagon (n=6-7) content were assessed by ELISA. Data is shown as mean values +/−SD, *p<0.05.

Article Snippet: Antibodies against full length APP (ab32136), BACE2 (ab8025), glucagon (ab10988), IDE (ab32216) and the insulin receptor (ab69508) were purchased from Abcam (Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of endocrinology

Article Title: Amyloid Precursor Protein in Pancreatic Islets

doi: 10.1530/JOE-17-0122

Figure Lengend Snippet: (A) Immunohistochemistry was performed on WT, APP−/−, and APP/PS1 mouse 2 month old 10μm pancreas cryosections using primary antibodies against IDE and BACE2 (n=7). Tissue was fixed with 4% paraformaldehyde and Vector VIP chromogen was used as the chromogen. (B) Immunohistochemistry was performed on 12 month old murine 10μm pancreas cryosections (n=4). Primary antibodies against amylin/IAPP, BACE2, CD68, Aβ oligomers (MOAB-2) and Aβ were utilized to detect proteins. Vector VIP was used as the chromogen.

Article Snippet: Antibodies against full length APP (ab32136), BACE2 (ab8025), glucagon (ab10988), IDE (ab32216) and the insulin receptor (ab69508) were purchased from Abcam (Cambridge, UK).

Techniques: Immunohistochemistry, Plasmid Preparation

Journal: The Journal of endocrinology

Article Title: Amyloid Precursor Protein in Pancreatic Islets

doi: 10.1530/JOE-17-0122

Figure Lengend Snippet: WT, APP−/−, and APP/PS1 pancreatic islets (n=3) were cultured overnight and the next day 50 islets from each animal were selected, pelleted, and lysed in RIPA buffer. Protein lysate was resolved by 10% SDS-PAGE then transferred to PVDF membranes and western blotted with antibodies against APP (Y188), IDE, GLUT4, and BACE2. Anti-GAPDH was used as a loading control. Optical density was measured for IDE and BACE2 and was normalized to WT optical density. Data is expressed as mean values +/−SD, *p<0.05.

Article Snippet: Antibodies against full length APP (ab32136), BACE2 (ab8025), glucagon (ab10988), IDE (ab32216) and the insulin receptor (ab69508) were purchased from Abcam (Cambridge, UK).

Techniques: Cell Culture, SDS Page, Western Blot, Control

Journal: The Journal of endocrinology

Article Title: Amyloid Precursor Protein in Pancreatic Islets

doi: 10.1530/JOE-17-0122

Figure Lengend Snippet: (A) Primary islet cultures from WT, APP−/−, and APP/PS1 mice were grown for 24 hours. Conditioned media was collected and protein content was assessed by western blot using antibodies against N-terminal APP. 22C11 detects both mouse and human APP while 6E10 is specific to human APP and detects sAPPα. Human islets were cultured for 24 hours and both conditioned media collected for analysis by western blot using anti N-terminal APP antibody (22C11). (B) Primary cultures of mouse islets were assessed for differences in insulin and glucagon secretion at 5mM glucose. Wild type islets were stimulated with 10 nM recombinant human sAPPα and supernatant was collected for insulin (n=8) and glucagon (n=4) ELISA. Insulin secretion data from multiple sAPP stimulation experiments were pooled by normalizing to the mean of the unstimulated control from each experiment. (C) The MIN6 insulin secreting cell line was grown as pseudo-islets and was stimulated with 0, 1nM, or 10nM recombinant sAPPα in GSIS assay at either 3.7mM or 16.7 mM glucose (n=6-8/condition). Data is expressed as the mean +/− SD, *p<0.05. (D) Human islets were treated with 10 nM recombinant sAPPα during the GSIS assay in 3.7, 10, and 16.7 mM glucose. After one hour of treatment, islet insulin secreted into the supernatant was assessed by ELISA. Data was pooled from five donors and normalized to basal insulin secretion at 3.7 mM. Data is expressed as the mean +/− SD, *p<0.05. 1μM forskolin was used with 16.7 mM glucose to potentiate insulin secretion as a positive control.

Article Snippet: Antibodies against full length APP (ab32136), BACE2 (ab8025), glucagon (ab10988), IDE (ab32216) and the insulin receptor (ab69508) were purchased from Abcam (Cambridge, UK).

Techniques: Western Blot, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Positive Control