Hrp conjugated goat anti rat igg

Whole cell extracts for SDS-PAGE were generated as follows (26 (link)): 1 × 10⁶ BMDCs or B cells were washed with ice-cold PBS and resuspended in 50 μl lysis buffer mixed with Na₃VO₄, NaF, and PMSF. Afterwards, protein concentrations were determined by the BCA protein assay described above. Sample proteins were boiled in the presence of a loading dye mixed with SDS and 2-ME for 10 min at 95°C. Then, 20 μg of total protein per sample were separated on a 12.5% polyacrylamide gel and afterwards transferred onto nitrocellulose filters (Schleicher & Schüll/GE Healthcare), with a pore size of 0.2 μm with the wet blotting device “Mini-Protean II Cell and System” (BioRad). Membranes were incubated with primary antibodies against human CD83 (1G11, kindly provided by Elisabeth Kremmer, LMU Munich, Germany), murine CD83 (R&D Systems) or Beta-actin (AC-74; Sigma-Aldrich), followed by HRP-conjugated goat anti-rat IgG (Cell Signaling Technology), donkey anti-goat IgG (Promega), or goat anti-mouse IgG (Promega) antibodies. Detection was performed with the chemiluminescent ECL Prime Western Blotting Detection Reagent (Amersham, GE Healthcare) on a high performance chemiluminescence film (GE Healthcare).

Cells were lysed with radioimmune precipitation assay (RIPA) buffer (Fujifilm Wako pure Chemical) containing Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The lysates were clarified through centrifugation at 15,000 rpm for 10 min, and the supernatant was collected. SDS-PAGE was performed on a 10% polyacrylamide gel for 50 min at 150 V. The samples were subsequently transferred to a poly(vinylidene difluoride) (PVDF) membrane (Bio-Rad, Hercules, CA, USA) using a Trans-Blot Turbo transfer system (Bio-Rad). The membrane was blocked with TBST buffer (20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween 20, pH 7.5) containing 5% BSA for 60 min at room temperature. The membrane was subsequently incubated overnight at 4°C with rat monoclonal MSR-A (clone: 7G5C33; BioLegend; 1:200 dilution) and rabbit polyclonal anti-β-actin (A2066; Merck; 1:1,000 dilution). The membrane was treated with horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (catalog number: 7077; Cell Signaling Technologies, Danvers, MA, USA; 1:1000 dilution) or goat anti-rabbit IgG (catalog number: 7074; Cell Signaling Technologies; 1:1000 dilution) for 60 min at room temperature. The membrane was treated using ImmunoStar Zeta (Fujifilm Wako Pure Chemical). Chemiluminescence images were acquired using an ImageQuantLAS500 imager (GE Healthcare, Chicago, IL, USA).

Primary antibodies: Monoclonal rat anti-AID (Active Motif, 39886); Polyclonal rabbit anti-mouse UNG (UNG 6103, custom made); Rabbit anti-human UNG (UNG PU059, made in-house (21 (link))); Monoclonal rabbit anti-MRPL11 (D68F2) XP (Cell Signaling 2066); Monoclonal mouse anti-β-Actin (ab8226); Polyclonal rabbit anti-GFP (ab290); Monoclonal rabbit anti-RPA2 [EPR2877Y] (ab76420): Polyclonal rabbit anti PCNA Ab (ab18197). Secondary antibodies: HRP-conjugated swine anti-rabbit IgG (Dako); HRP-conjugated goat anti-mouse IgG (Dako); HRP-conjugated goat anti-rat IgG (Cell Signaling); IRDye 800CW Goat anti-rabbit (LI-COR); IRDye 680RD goat anti-rabbit (Li-COR); IRDye 680RD goat anti-mouse (Li-COR).

Total mouse brain proteins were extracted using Western-IP Lysis Buffer (Beyotime,Wuhan, China) and quantified using the BCA protein assay kit (Beyotime, Wuhan, China). In total, 10 μg of total proteins were electrophoresed on 12% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with PBS/Tween 20® (PBST) containing 5% non-fat milk for 90 min at room temperature and then incubated overnight at 4 °C with the following primary antibodies: anti-caspase-3, anti-cleaved caspase-3, anti-caspase-6, anti-cleaved caspase-6, anti-RIP3, anti-Beclin-1 (Cell Signaling Technology, Danvers, USA), anti-caspase-4, anti-phosphor-RIP3 (Abcam, Cambridge, UK), anti-LC3B (Beyotime,Wuhan, China), and anti-β-actin (Cell Signaling Technology, Danvers, USA) as the control. After incubation with HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-rat IgG (Cell Signaling Technology), the nitrocellulose membranes were washed three times, and bands were visualized by chemiluminescence with Pierce Enhanced Chemiluminescence Detection Reagent (Millipore, Burlington, USA) on the ChemiDoc™ Touch imaging system (Bio-Rad, Hercules, USA). Relative band intensity was analysed by AlphaViewTM software (Alpha Innotech Corporation, San Jose, USA).