Fusion fx5 image analysis system

All cells with or without treated with chemicals were lysed in RIPA buffer (50 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxylcholate) including protease inhibitors (Roche, Indianapolis, USA). Protein concentrations were measured using a BCA Protein Assay Kit (Dingguo, Beijing, China). Equal amounts of protein extracts were separated on a 12% SDS-PAGE gel and then transferred onto PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked by 5% nonfat dry milk in TBST (25 mM Tris, pH 7.4, 1.5 M NaCl, and 0.05% Tween-20) for 1 h at RT and probed with primary antibodies against furin (Abcam, Cambridge, UK), CEBPβ (Cell Signaling Technology, Danvers, MA, USA), GATA1 (Cell Signaling Technology), and GAPDH (Proteintech, Wuhan, Chian) overnight at 4°C. The blots were washed and incubated for 1 h with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Proteintech). The bands were visualized using an ECL reagent (Advansta Inc., Menlo Park, CA, USA) and a Fusion FX5 image analysis system (Vilber Lourmat, Marne-la-Vallée, France). Relative protein expression levels were calculated using the Quantity One software (Bio-Rad) with normalization to the GAPDH signal.

Brain tissues were homogenized and sonicated in ice‐cold lysis buffer [50 mM Tris–HCl, pH 8.0, 140 mM NaCl; 1.5 mM MgCl₂; 0.5% NP‐40 with complete protease inhibitor cocktail (Roche)]. Protein concentration was measured in the supernatant by BCA Protein Assay (Dingguo). The monoclonal or polyclonal antibodies used were BACE1 (Abcam, 1:1000); APP full length (A8717, Sigma, 1:3000); sAPPα (6E10, Covance, 1:1000); sAPPβ (Covance, 1:500); ADAM10 (Abcam, 1:1000); PSEN1 (Proteintech, 1:500); GAPDH (Proteintech, 1:10000); tau46 (CST, 1:1000); phosphorylation‐tau 181 (CST, 1:1000); phosphorylation‐tau 202 (Abcam, 1:5000); and phosphorylation‐tau 396 (Abcam, 1:10000). The secondary antibodies were goat anti‐rabbit or anti‐mouse horseradish peroxidase‐labeled antibodies (Proteintech, 1:5000). The membranes were visualized using an ECL reagent (Thermo) and a Fusion FX5 image analysis system (VilberLourmat). Relative protein intensities were calculated using Quantity One software (Bio‐Rad).

Electrophoresis gel, electrophoresis solution, electrophoresis solution, and tris-buffered saline with 0.1% Tween (TBST) were prepared prior. BCA was used to determine protein concentration, and each well was loaded equally. The gels were transferred to a 0.45-µm PVDF membrane (A10178785, GE, USA); 5% nonfat milk in TBST was used to block the membrane for 1 h at room temperature. The membranes were then incubated at 4°C overnight with primary antibodies for 16 h. The primary antibodies were as follows: anti-RGMa (1:10,000, ab169761, Abcam, USA); anti-BMP2 (1:1,000, YT5651, Immunoway, USA); anti-BMPR II (1:500, 220550, ZEN Bio, Chengdu); anti-YAP (1:1,000, 14074, Cell Signaling Technology, USA); anti-ZO-1(1:1,000, 40-2200, ThermoFisher Scientific, USA); anti-GAPDH (1:10,000, 20494-1-AP, Proteintech, Wuhan); and anti-beta Tubulin (1:4,000; 10068-1-AP, Proteintech, Wuhan). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, SA-00001-2, Proteintech; goat anti-mouse IgG, SA00001-1, Proteintech) for 1 h at room temperature. Images were captured by the Fusion FX5 image analysis system (Vilber Lourmat, F77601 Marne-la-Vallée cedex 3, France).

We selectively mated 6- to 8-week-old female and male mice (C57BL/6J), isolated [20 (link)] cortical tissue from embryonic and postnatal mice at different developmental time points (E14.5, E17.5, P0, P7, P14, P21, and P60), and extracted total protein samples for Western blotting experiments. Protein samples were separated by 12.5% SDS-polyacrylamide gel electrophoresis, and the gels were separated and electrically transferred to 0.22-μm PVDF membranes. The membranes were blocked with 10% skim milk powder containing tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature and then incubated with the noted CXCR5 and CXCL13 antibody/β-tubulin antibody overnight at 4°C. The membranes were washed 3 times with TBST for 10 min each and then incubated in anti-mouse or anti-rabbit secondary antibody containing horseradish peroxidase at room temperature for 1 h. The membranes were then washed three times with TBST and tagged using enhanced chemiluminescence reagents (Thermo Fisher, MA), and a Fusion FX5 image analysis system (Vilber Lourmat, Marne-la-Vallée, France) was used to visualize the bands. Relative protein levels were determined by normalizing the signals for CXCR5 to β-tubulin levels and CXCL13 to β-tubulin and CXCR5, p-Cofilin, Cofilin to actin, and p-Cofilin / Cofilin and F-actin/G-actin using Quantity One software (Bio-Rad, CA).