Astrocytic PAD2 governs microglia activity to exacerbate amyloid pathology via citrullinated vimentin.Zhang et al.

Description

This dataset contain all uncropped original Western blot images, retaining molecular weight markers and full membrane backgrounds for antibody specificity verification and band quantification.

Steps to reproduce

Frozen hippocampus were homogenized with RIPA lysis buffer (CST, 9806) containing a cocktail of protease inhibitors (MCE,HYK0010)and PMSF (CST, 8553)，and sonicated at 100 W (1 s on and 2 s off) for 1 min on ice and then lysed on ice for 30 min, followed by centrifugation at 12,000 g for 20 min at 4℃. The lysates were then centrifuged and the supernatants collected. The protein concentration of lysates was measured by BCA assays (Beyotime, P0009) and was adjusted to the same final concentration. After heat denaturation, equal amounts of protein in the lysates were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane（Millipore，IPVH0000). The membranes were blocked with 5% bovine serum albumin (BSA) in tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature followed by incubation with the indicated primary antibodies at 4℃ overnight. After three washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The immunoreactive proteins were then detected using an enhanced chemiluminescent (ECL) substrate (MCE, HY-K1005) and visualized with Chemiluminescence Imager-eBLOT. The following primary antibodies were used: PAD2 (Proteintech, 12110- 1-AP ), Pan-cit Modified Citrulline antibody C4 (sigma, MABS487), Cit-vimentin (customize from Jingjie, Hangzhou, China), Vimentin (Proteintech,10366-1-AP ), Iba1 (Novus, NB100-1028), GFAP (SY5Y, 173011), PSD95 (abcam, AB18258), Synaptophysin (Cell Signaling Technology, 9020S), anti-APP (millipore, 171610), β-actin (abcam, AB8226), α-Tubulin (proteintech, 66031-1), GAPDH (proteinrech, 60004-1-AP) and Cofilin (Proteintech, 10960-1-AP ). The band intensities were quantified with ImageJ software, and the number inserted below the band indicates the (average) ratio for protein or modification with reference to the loading control.

Institutions

• Fujian Medical University

  Fujian, Fuzhou

Categories

Funders

• National Natural Science Foundation

  Grant ID: U22A20298
• National Natural Science Foundation of China

  Grant ID: 82271468
• National Natural Science Foundation

  Grant ID: 82430041

Licence