Complete tablets mini

Proteins were isolated from retinal or choroid tissue of p17 and p31 Vldlr^−/− and control mice (n = 5 per group) using RIPA buffer (R0278, Sigma-Aldrich) containing protease (Complete Tablets Mini, 0463159001, Roche, Basel, Switzerland) and phosphatase inhibitors (Phosstop, 04906845001, Roche). The amount of isolated protein was measured using a colorimetric assay (Pierce™ BCA Protein Assay Kit, 23225, Thermo Fisher Scientific). To examine cytokine concentrations in retinal samples, we used a multiplex electrochemiluminescence panel (R-Plex, Meso-Scale Discovery, Rockville, MD, USA) according to the manufacturer’s instructions. This kit simultaneously measured the protein levels of CC-chemokine ligand (CCL12) and Vascular Endothelial Growth Factor A (VEGFA) in the same samples. These factors were chosen based on the RNA Seq results of this study and represented relevant or differentially expressed genes in RAP microglia compared to steady-state microglia. For statistical analysis, all values below the detection limit were assigned to half of the respective values of the detection limit.

Cardiac muscle strips of 2.69 ± 0.82 mm length, 0.32 ± 0.08 mm width, and 0.20 ± 0.09 mm² cross‐sectional area (CSA), calculated by 2πr² assuming a circular shape, were isolated from human cardiac tissues and EHTs. For contractile measurements, strips were permeabilized in a pCa9 EGTA‐buffer containing 1% Triton X‐100 at 4°C for 18 as described previously (Kooij et al, 2010; Stoehr et al, 2014; Friedrich et al, 2016). The next day strips were either used directly for measurements or stored at −20°C in a 50% glycerol/relaxing solution with protease inhibitors (EDTA‐free, complete tablets, mini, Roche). Measurement of contractile function was performed using a fiber test system (1400A; Aurora Scientific) as reported previously (Friedrich et al, 2016; Stucker et al, 2017). A Hill equation was fitted to the data points (Hill et al, 1980) to estimate pCa₅₀ as the free Ca²⁺ concentration yielding 50% of the maximal force and nH representing the Hill coefficient. The pCa₅₀ represents the parameter of myofilament Ca²⁺ sensitivity.

PathScan^® Intracellular Signaling Array Kit (Chemiluminescent Readout, cat. Number 7323, Cell Signaling Technology) was used for this purpose. All steps were done in accordance to the manufacturer’s protocol at RT. Briefly, cells were cultured for 3 days in different substrates and then washed with ice-cold PBS. Whole protein lysates were prepared using ice-cold 1X Cell Lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors (complete Tablets Mini and PhosSTOP EASYpack, Roche). The Array Blocking Buffer was added and incubated for 15 min on an orbital shaker. The protein lysate was added and incubated for 2 h. After washing, the Detection Antibody Cocktail was added and incubated for 1 h on an orbital shaker. HRP-linked streptavidin was added and incubated for 30 min. LumiGLO^®/Peroxidase reagent was added and images captured immediately after, using a Chemidoc (chemiDoc™ XRS+, BioRad) and quantified using ImageLab 5.0 Software.

Yeast culture was grown in YES to OD₅₉₅ = 0.3–0.5. 45 mL of culture was incubated with 1% of formaldehyde for 20 min for cross-linking. The reaction was quenched with 125 mM glycine. Cells were lysed with lysis buffer (50 mM HEPEs-KOH, pH 7.5, 140 mM sodium chloride, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA) supplemented with protease inhibitors (cOmplete Tablets Mini, EASYpack; Roche) by vortexing with glass beads (0.5 mm; Biospec Products). Chromatin was sheared to 500–1,000 bp by sonication (amplitude, 100%; process time, 5 min; ON time, 30 s; OFF time, 2 min). Chromatin lysate was incubated with specific antibodies (anti-H3K36me2 Active Motif 39255; antiH3K36me3 Active Motif 61101; Myc-tag Santa Cruz 9E10: sc-40) at 4°C overnight rotating. Chromatin was pulled down with cold 50% protein A-Sepharose beads in lysis buffer (Sigma; P3391) for 3 hr and washed six times with washing buffer (50 mM Tris-HCl, 1% Triton X-100, 150 mM sodium chloride, 5 mM EDTA, 0.5% NP-40). De-cross-linking was performed with 10% Chelex in ultra-pure water and boiling for 10 min. DNA enrichment was established by qPCR with SYBR green according to the manufacturer’s recommendations and percentage of the whole-cell extract method. Primers used in this study are listed in Table S2.

Primary antibodies p38 MAPK (8690S), Phospho-p38 MAPK (9216S), p44/42 MAPK (Erk1/2) (3A7) (9107S), Phospho-p44/42MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (4370S), SAPK/JNK (9252), Phospho-SAPK/JNK (Thr183/Tyr185) (G9) (9255S), Akt (pan) (40D4) (2920S), Phospho-Akt (Ser473) (D9E) (4060S) and MyD88 (D80F5) (4283) were purchased from Cell Signaling and TLR4 (MA5-16216) from Thermo Fisher Scientific, Waltham, MA, USA. Secondary antibodies StarBright Blue 700 (12004158) and StarBright Blue 520 (12005869) were purchased from Bio-Rad, Hercules, CA, USA. For flow cytometry APC, FITC and PE-labeled antibodies were purchased from eBiosciences, San Diego, CA, USA (FITC-labeled anti-human CD34 (11-0349-42), APC-labeled anti-human CD45 (17-0459-42), APC-labeled anti-human CD73 (17-0739-42), FITC-labeled anti-human CD90 (11-0909-42), PE-labeled anti-human CD105 (12-1057-42)). For Western blotting, RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) complemented with cOmplete Tablets Mini and PhosStop (Roche, Basel, Switzerland), Neutralization monoclonal blocking antibody TLR-4 (HTA125) (14-9917-82) 5 μg/mL from Thermo Fisher Scientific, Recombinant Human HMGB1 Protein (1690-HMB) R&D Systems was used.

Western blotting was carried out as previously described^{70 (link)}. Overall, lysates were prepared in RIPA buffer (Sigma) supplemented with protease (cOmplete Tablets, Mini, EDTA-free, Roche) and phosphatase (PhosSTOP, Roche) inhibitors. Protein lysates were loaded with 4× Laemmli sample buffer (Bio-Rad) and 2-mercaptoethanol (Bio-Rad) and run by SDS-PAGE, and were subsequently transferred to PVDF membranes (Bio-Rad) using Trans-Blot Turbo transfer buffer (Bio-Rad) and a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% blotting-grade blocker non-fat dry milk (Bio-Rad), followed by overnight 4 °C incubation with the appropriate primary antibody (Supplementary Table 3), and 1 h room temperature incubation with an anti-rabbit or anti-mouse IgG (H + L)-HRP conjugate (Bio-Rad) secondary antibody. Blots were imaged using chemiluminescent substrate (Thermo Scientific) and a LAS-3000 imager (Fujifilm) or ChemiDoc Imaging System (Bio-Rad). When necessary, blots were stripped with Restore PLUS western blot stripping buffer (Thermo Scientific), followed by repeat blocking and antibody incubations. Uncropped images of the western blots from Fig. 5a are presented in Supplementary Figure 14.