Gel pro analyzer version 3

The proteins were extracted with buffer containing 1 M PBS, 5 Mm EDTA, and 0.5% Triton X-100. After centrifugation (13,000 rpm for 10 min, 4 °C), the supernatant was collected for Western blot analyses. Protein (20 µg/lane) was electrophoresed on 10–15% SDS gel and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature (RT) and then incubated with primary antibodies against α-tubulin, Bcl2, Bax, cleaved caspase-3, p-p38, p-JNK, and p-p53 (1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, and 1:1000, respectively, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. The membranes were incubated with HRP-conjugated anti-rabbit IgG secondary antibodies (1:2000, Abfrontier Co., Ltd., Seoul, Republic of Korea) and HRP-conjugated anti-mouse IgG secondary antibodies (1:2000, Abfrontier Co., Ltd., Seoul, Republic of Korea) for 2 h at RT. The protein bands were visualized using a chemiluminescence detection kit (Thermo Scientific, South Logan, UT, USA). The same membranes were subsequently used for α-tubulin immune detection, and equal protein loading was ensured. The optical density for quantification was obtained using Gel-Pro Analyzer version 3.1 (Media Cybernetics, Silver Spring, MD, USA).

For protein extraction, hippocampus tissue samples were thawed and homogenized in lysis buffer containing 40 mg/mL CHAPS, 50 mM Tris-HCl, pH 7.6, 0.4808 mg/mL urea, 1 mM phenylmethylsulfonyl fluoride, 5 μg/mL aprotinin (Amresco, USA), and 5 μg/mL leupeptin (Amresco). Lysates were centrifuged at 14,000 g for 5 min at 4°C, and the supernatants were collected. Supernatant proteins (30 or 60 μg/lane) were separated on 10% SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes (Invitrogen, USA). Membranes were blocked in 8% skim milk for 1 h at RT, then incubated in anti-DSCAM (1:1,000; N-16, sc-79437; Santa Cruz) overnight at 4°C and anti-β-actin as an internal reference (1:1,000; sc-81178; Santa Cruz). Immunolabeled membranes were then incubated in HRP-labeled goat anti-rabbit IgG (1:4,000; Zymed) for 45 min at RT. Bands were visualized using enhanced chemiluminescence and autoradiography (Hyperfilm-ECL; Amersham, Sweden). We analyzed band optical density using an image analysis system (Gel-Pro Analyzer Version 3.0, Media Cybernetics Inc., USA) and calculated the ratio of DSCAM to β-actin optical density for estimation of DSCAM expression.

Primers for genus Clostridium, Bifidobacterium, Lactobacillus, Klebsiella, and species E. coli and L. plantarum were designed as per previous literature [45 ,46 (link),47 (link),48 (link)]. The primers used in this study are detailed previously [49 (link)]. All known bacteria were identified using a Universal primer. The PCR amplification was carried out by adding the DNA extracted (as template) to a PCR premixture containing nuclease-free water, PCR buffer, Taq polymerase, dNTPs, and primer. PCR conditions were set as previously optimized [49 (link)]. The PCR products were quantified using gel electrophoresis with ethidium bromide. Visualized in Gel-Pro analyzer version 3.0 (Media Cybernetics LP, Rockville, MD, USA) [47 (link)].

The following primers were used: Bifidobacterium, Lactobacillus, Escherichia coli, Clostridium, and 16S rDNA (universal) according to what was previously described [39 (link),40 (link),41 (link)]. Essentially, each bacterial group is reported as a relative proportion of the bacteria to the universal primer. PCR products were applied to 1.5% agarose gel with ethidium bromide stain and quantified with Gel-Pro analyzer version 3.0 (Media Cybernetics LP, Rockville, MD, USA).

Bifidobacterium, Lactobacillus, Escherichia coli, Clostridium, Klebsiella, and L. plantarum primers were used. 16S rRNA was the universal primer and internal standard. Therefore, the proportions of each bacterial group are presented. PCR products were applied to 1.5% agarose gel with ethidium bromide stain and quantified with Gel-Pro analyzer version 3.0 (Media Cybernetics LP, Rockville, MD, USA).