Goat anti rabbit hrp

Total protein was extracted from cultured HT22 cells using protein lysis buffer (Beyotime Biotech, China). The protein concentration was determined with a BCA kit (P0010S, Beyotime Biotech). The proteins in each sample were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Western blot analysis was carried out using the following primary antibodies raised against target proteins: rabbit anti-SIRT3 (dilution 1:1000, ab189860, Abcam), rabbit anti-SOD2 (dilution 1:1000, ab68155, Abcam), rabbit anti-acetyl-SOD2 (dilution 1:1000, AF3751, Affinity Biosciences, Melbourne, Victoria, Australia), rabbit anti-cleaved caspase 3 (dilution 1:1000, 19677-1-AP, Proteintech, Fisher Scientific), and rabbit anti-β-actin (dilution 1:1000, ab181602, Abcam) antibodies overnight at 4 °C. The following secondary horseradish peroxidase (HRP)-conjugated antibodies were used at 1:5000 dilution: goat anti-rabbit HRP (Millipore, Watford, UK). Blots were visualized with enhanced chemiluminescence detection reagents using a Chemidoc TM Touch Imaging System (Bio-Rad, Hercules, CA).

Total protein was extracted from hippocampal tissues with protein lysis buffer (Beyotime Biotech, China). The protein concentration was determined with a BCA kit (P0010S, Beyotime Biotech). The proteins in each sample were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Western blot analysis was carried out using the following primary antibodies raised against target proteins: rabbit anti-acetyl lysine (dilution 1:1000, ab190479, Abcam, Cambridge, UK), rabbit anti-Sirt3 (dilution 1:1000, ab189860, Abcam), rabbit anti-SOD2 (dilution 1:1000, ab68155, Abcam), rabbit anti-acetyl-SOD2 (dilution 1:1000, AF3751, Affinity Biosciences, China), and rabbit anti-β-actin (dilution 1:1000, ab181602, Abcam) antibodies overnight at 4 °C. The following secondary horseradish peroxidase (HRP)-conjugated antibodies were used at 1:5000 dilution: goat anti-rabbit HRP (12–348, Millipore, Watford, UK) and goat anti-mouse HRP (sc-2005, Santa Cruz Biotechnology, Dallas, TX, UK). Blots were visualized with enhanced chemiluminescence detection reagents using a Chemidoc TM Touch Imaging System (Bio-Rad, Hercules, CA, USA).

DiC8-PIP₃, In(1,3,4,5)P₄, In(1,4,5)P₃ (IP₃), and GNF362 were obtained from Cambridge Bioscience Ltd. Fura2-AM was obtained from Thermo Fisher Scientific, and U46619 was obtained from Tocris. The phospho-AKT antibody recognizing phospho-Ser⁴⁷³ (clone 11E61) was obtained from Upstate. The pan-AKT antibody (C67E7) recognizing Akt1, Akt2, and Akt3 proteins was obtained from Cell Signaling Technology. Goat antimouse antibody linked to horse-radish peroxide (HRP) and Goat antirabbit-HRP were purchased from Millipore. Antibodies to GAP1(IP4BP/RASA3), ITPKB, ITPKA, and BTK were purchased from (Santa Cruz Biotechnology Inc). The anti-Rap1(A+B) and IP₃-ELISA kit was purchased from Abcam Ltd. GTPγS and RalGDS-RBD beads were obtained from Abcam Ltd. PI(3,4,5)P₃ PIP beads (PIP₃ beads) were obtained from Echelon Biosciences (via 2B Scientific). Vena8 Cellix microfluidic chip channels were obtained from Cellix Ltd.

Antibodies against rRBP2-P1 were produced by Thermo Fisher Scientific (USA). Briefly, two rabbits were immunized intramuscularly with 0.50 mg of rRBP2-P1 emulsified with Freund’s complete adjuvant on day 0, followed by three boosts of the antigen emulsified with incomplete Freund’s adjuvant on days 14, 42, and 56. The serum was collected on day 70. Serum was also used for rabbit IgG purification by protein G Sepharose (GE Healthcare), according to the standard protocol. The specificity of serum for rRBP2-P1 was confirmed by Western blotting against recombinant P. vivax RBPs (rRBPs) (13 (link)). For this, 1 ng of each rRBP was separated on SDS-PAGE gels and transferred to the membrane. The membrane containing proteins was probed with rabbit anti-rRBP2-P1 IgG (1 μg/ml), followed by goat anti-rabbit HRP (0.2 μg/ml; Millipore). Reactions were detected with chemiluminescence. Coomassie-stained SDS-PAGE of the same rRBP samples (1 μg/lane) was used to demonstrate that each sample contained a protein (see Fig. S1 in the supplemental material).

Brains were dissected and immediately fixed in 5 volumes of PLP fixative (4% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate; pH 7.4) at 4°C overnight and cryoprotected using 10% and 20% glycerol/2% dimethylsulfoxide, in 0.1 M PBS, pH 7.3 (24 h each). Serial, frozen sections (40 µm, coronal) were cut with a sliding microtome from the anterior frontal pole of to the caudal occipital region. All sections intended to be subjects of comparative analyses were processed together and incubated for the same time periods in all of the reagents. For Aβ42 immunohistochemistry, sections were treated with>95% formic acid (Sigma) for 2 min with gentle agitation, washed with PBS, and then transferred to a solution of PBS/10% Goat Serum (Gibco) for 1 h at room temperature (RT). Sections were probed with rabbit anti-Aβ42 (1∶2500, Invitrogen) overnight at RT in 0.3% triton-X 100, 2% Goat Serum (Gibco), 0.008% sodium azide PBS. Following washing, sections were probed with goat anti-Rabbit-HRP (Millipore, 1∶1000) in a solution of 2% Goat Serum/PBS for 3 h at RT. After incubation in a developing solution containing diaminobenzidine (DAB), sodium imidazole, and hydrogen peroxide, sections were mounted on subbed slides. Photomicrographs were taken with 2X and 4X magnification objectives and images of the hippocampus were analyzed with ImageJ software (NIH).