Journal: Human Molecular Genetics

Article Title: Cross-species genetic screens to identify kinase targets for APP reduction in Alzheimer's disease

doi: 10.1093/hmg/ddz034

Figure Lengend Snippet: Chronic knockdown of PKCβ does not cause compensatory changes in GSK3β or in APP phosphorylation at Thr668. (A,B) Western blots for total GSK3β and GSK3β phosphorylated at serine 9 (A) and for total APP and APP phosphorylated at threonine 668 (B) in cortical homogenates from mice harvested 6 months after P0 AAV injection with scramble or shPKCβ. Note that the same samples were probed for APP and PKCβ in Figure 6A. (C) Quantification of total GSK3β relative to GAPDH. Values for all graphs were normalized to the respective scramble control groups. Student’s t-test, P = 0.82. (D) Quantification of serine 9-phosphorylated GSK3β relative to total GSK3β. Student’s t-test, P = 0.35. (E) Quantification of total APP (detected with antibody 22C11) relative to GAPDH. Student’s t-test, P = 0.96. (F) Quantification of threonine 668-phosphorylated APP relative to total APP. Student’s t-test, P = 0.80.

Article Snippet: Membranes were blocked in TBS containing 0.1% Tween-20 and 5% non-fat dry milk for 1 hr at RT and probed overnight at 4°C with primary antibody diluted in blocking solution: 6E10 (Biolegend, San Diego, CA, #SIG-39320, 1:5000), Y188 (Abcam, Cambridge, UK, #ab32136, 1:5000), 22C11 (Millipore, #MAB348, 1:3000), phospho-APP Thr668 (Cell Signaling, #6986, 1:1000), GSK β (Cell Signaling #12456, 1:1000), phospho-GSK3 β Ser9 (Cell Signaling #9322, 1:1000), PKC β II (ThermoFisher #PA5–13741, 1:250), PKC α (Abcam #ab32376, 1:5000) or GAPDH (Millipore #AB2302, 1:10,000).

Techniques: Western Blot, Injection

Journal: International Journal of Molecular Sciences

Article Title: Systemic Exposure to Air Pollution Induces Oxidative Stress and Inflammation in Mouse Brain, Contributing to Neurodegeneration Onset

doi: 10.3390/ijms21103699

Figure Lengend Snippet: Amyloidogenic precursor protein (APP) processing analysis after single and repeated instillations of BB and DEP. Representative immunoblotting images of amyloid precursor protein (APP), phosphorylated APP on threonine 668 (p-APP Thr668 ), and beta-secretase 1 (BACE1) analysis in mice after single ( A ) and repeated ( E ) instillations with 50 µg of BB or DEP/100 µL 0.9% NaCl. Histograms display p-APP Thr668 /APP, APP, and BACE1 protein levels in mice after single ( B – D ) and repeated ( F – H ) instillations with BB and DEP, with respect to sham. Proteins are normalized to corresponding total proteins revealed by Ponceau in each lane , and the data are expressed as means ± SEM ( n = 6). Statistical differences were tested accordingly by one-way ANOVA followed by Tukey post hoc comparison. ** p < 0.01 vs. sham mice; **** p < 0.0001 vs. sham mice; § p < 0.05 vs. BB-treated mice.

Article Snippet: Immunoblottings were performed using anti-rabbit polyclonal APP (CT695) (1:500) (Thermofisher Scientific™ Cat# 51-2700, RRID:AB_2533902), anti-rabbit monoclonal phospho Thr668 APP (p-APP Thr668 ) (1:1000) (Cell Signaling Technology Cat# 6986, RRID:AB_10831197), anti-rabbit monoclonal BACE1 [EPR3956] (1:2000) (Abcam Cat# ab108394, RRID:AB_10861218), anti-rabbit polyclonal cyclooxygenase-2 (COX-2) (1:1000) (Cell Signaling Technology Cat# 4842, RRID:AB_2084968), anti-rabbit polyclonal cytochrome 1b1 (Cyp1b1) (1:800) (Santa Cruz Biotechnology Cat# sc-32882, RRID:AB_2089112), anti-rabbit polyclonal heme oxygenase-1 (HO-1) (1:200) (Santa Cruz Biotechnology Cat# sc-10789, RRID:AB_648281), anti-goat polyclonal Hsp70 (1:200) (Santa Cruz Biotechnology Cat# sc-1060, RRID:AB_631685), and anti-rabbit polyclonal inducible nitric oxide synthase (iNOS) (1:400) (Santa Cruz Biotechnology Cat# sc-8310, RRID:AB_2152867) antibodies.

Techniques: Western Blot