Tgx sds page gel

Dissected aortas were homogenized in lysis buffer using glass grinder and pestle 20 as previously described [68 (link)]. After centrifugation, protein levels were determined in supernatants using DC protein assay kit (BioRad, Hercules, CA). For each sample, 50 μg protein was loaded on 7.5% or 10% TGX SDS-PAGE gels (BioRad). Antibodies against APP (1:250; cat# 51-2700, Invitrogen, Carlsbad, CA), BACE1 (1:250; cat# S5606, Cell signaling, Danvers, MA), ADAM10 (1:500; cat# AB19026, Millipore, Burlington, MA), eNOS (1:250, cat# 610297, BD Biosciences, San Jose, CA), inducible nitric oxide synthase (iNOS; 1:250, cat# 610333, BD Biosciences), COX1 (1:500; cat# 35-8100, Invitrogen), and COX2 (1:250; cat# 610204, BD Biosciences) were used. The antibody specificities for APP, BACE1, and eNOS were verified in knockout mice obtained from The Jackson Laboratory (stock #004133, stock #004714, and stock #002684, respectively; Supplementary Figure 5). In addition, C57BL/6J mouse heart was used as positive control for COX1, and whole cell lysate RAW 264.7 (cat# ab7187, Abcam) was used to identify COX2 and ADAM10 bands (Supplementary Figure 6). All blots were reprobed with β-actin antibody (1:50,000, A5316, Sigma). Densitometry analyses were performed in Odyssey Fc imaging system with Image Studio^™ 5.2 software (Li-Cor Biotechnology, Lincoln, NE).

The expression of PLA2R1 in HEK293 cells overexpressing PLA2R1 with a Tetracycline-Regulated Expression System (PLA2R1/HEK293 T-REx) after induction with tetracycline was analyzed by SDS-PAGE under nonreducing conditions. A control without induction with tetracycline was performed. Total proteins (10-50 μg/well) were run on 4-15% precast TGX SDS-PAGE gels (Bio-Rad) and transferred to methanol-soaked PVDF membranes (Bio-Rad) under semidry conditions using Trans-blot Turbo (Bio-Rad) at 25 V constant for 12 min. Membranes were blocked overnight at 4°C in 5% milk with PBS-Tween (PBS-T) 0.05% and then incubated with primary and secondary antibodies for 2 h at room temperature. Primary antibodies were diluted with 0.5% dry milk in PBS-T. Membranes were prepared in replicates and probed with a serum of patient with anti-PLA2R1 antibodies diluted at 1 : 100. Secondary antibody for iMN sera was HRP-conjugated mouse anti-human IgG (Southern Biotech #9200-05) diluted 1 : 30,000 in PBS-T. Membranes were washed three times for 5 min in PBS-T after incubation with primary and secondary antibodies. Detection of protein bands was performed with a chemiluminescent substrate (Millipore) and a Fuji LAS3000 imager.

S-30 extracts were prepared from S. aureus JE2 by cryomilling cell disruption (see “Ribosome profile analysis” below). A runoff reaction was performed by incubating the lysate at 25°C for 70 min with 0.15 volume of runoff premix (0.75 M HEPES [pH 7.5], 7.5 mM dithiothreitol [DTT], 21.3 mM magnesium acetate, 75 μM twenty l-amino acids, 6 mM ATP, 20 mg/ml phosphoenolpyruvate, 50 U pyruvate kinase) relative to the volume of lysate input. The extracts were then dialyzed in Slide-A-Lyzer cassettes (Thermo Fisher) against three changes of buffer A (20 mM HEPES [pH 7.5], 14 mM magnesium acetate, 100 mM potassium acetate, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF]), centrifuged at 4°C at 20,800 × g for 10 min, and stored at −80°C.
Linear DNA fragments containing the hpf promoter fused to a gfp or a luc reporter were PCR amplified with P630/P631 (Table 1) using pLI50gfp or pLI50luc as a template. Typical 25-μl reaction mixtures contained 500 ng of DNA template, 10 μl of translation premix (81 (link)), 2.5 μl of 1 mM l-amino acids lacking methionine, 7.5 μl of S-30 extract, 200 ng/μl anti-ssrA oligonucleotide (5′-TTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCGAGTA-3′), and 10 μCi Tran³⁵S-label (MP Biomedicals). After a 1-h incubation at 37°C, protein samples were precipitated in 4 volumes of acetone, resolved on 4% to 20% TGX SDS-PAGE gels (Bio-Rad), and autoradiographed.

We used western analysis to investigate the cleavage of Immp2l substrates Cyc1 and Gpd2. Mouse tissues and mitochondria purified from these tissues (see section on Mitochondrial isolation) were freshly prepared in RIPA Buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing a phosphatase inhibitor cocktail (Halt #78429 Thermofisher Scientific 1:100) before adding 1:1 2× Laemmli buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.002% bromophenol blue, 0.0625 M Tris HCl). Protein samples (30 µg) were heated at 95 °C for 5 min before electrophoresis on precast TGX SDS-PAGE gels (Bio-Rad, Hercules, CA, USA) followed by Western transfer to 0.2 µM PDF membranes according to established protocols [72 (link)]. Membranes were probed with rabbit polyclonal antibodies against mouse proteins: Cyc1 (#10242-1-AP ProteinTech at 1:1000), Gpd2 (#17219-1-AP Proteintech at 1:500), Aifm (#VPA000017KT from Bio-Rad at 1:1000), Smac (#10434-1-AP Proteintech at 1:500), β-Actin (#20536-1-AP Proteintech at 1:3500) and Total OXPHOS Rodent WB antibody cocktail (#ab110413 Abcam at 1:250). Goat anti-rabbit horseradish peroxidase-conjugated secondary (#SA00001-2 Proteintech at 1:2000) was used with Luminol chemiluminescent reagent (Bio-Rad) to visualize proteins using the LI-COR model 2800 imaging system.

E. coli BL21(DE3)pLysS cells were co-transformed with pMBP-Parallel1-PupR CCSSD and pET41-GST-PupB NTSD. Co-transformants were selected by growing on LB agar medium containing 100 μg/ml of ampicillin and 15 μg/ml of kanamycin. Co-expression followed the same purification procedure as for the individual proteins. Harvested cells were lysed and cell debris pelleted by centrifugation. The clarified supernatant was divided into two equal aliquots and combined with either 5 ml of amylose resin or 5 ml of GSH-Sepharose resin. The columns were incubated for 30 min at 4 °C. Each column was washed with 10 column volumes of lysis buffer, then eluted with lysis buffer + 20 mm maltose or lysis buffer + 15 mm GSH as appropriate. Total protein content was determined by Bradford assay, and 20 μg of protein were loaded onto a 4–20% TGX SDS-PAGE gel (Bio-Rad). Gels were stained with Coomassie Blue and qualitatively analyzed for protein association. This protocol was repeated for all pulldown analyses. The identity of the proteins in the pulldown assays was confirmed by Western blotting, using commercially available anti-MBP-HRP (New England Biolabs) or anti-GST-HRP (GE Healthcare) antibodies.

For protein analysis, samples were divided into methylation high, methylation intermediate, and methylation low groups based on the beta-values across MAP2K3 DMR1 and samples from each group were randomly chosen as a representative subset of the discovery cohort. Protein lysates were prepared by manually homogenizing frozen tissue in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Proteins were separated on a 4–20% TGX SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane (Invitrogen). Blots were blocked in 5% dry milk in TBST buffer (20 mmol/L Tris–HCl pH 7.4, 150 mmol/L NaCl, 0.1% Tween-20) and incubated at 4 °C overnight in primary antibody; MKK3 (Cell Signaling #5674), p38 (Cell Signaling #9219), phospho-p38 (Cell Signaling # 4511), phospho-ERK1/2 (Cell Signaling #9101), and β-Actin (Cell Signaling #3700). Densitometry was done in ImageJ.

Strains IF235 (sir2Δ), IF140 and IF230 (sir2 over-expression; sir2OE) were grown in synthetic complete media (Sunrise Scientific) to OD 1.0, and whole cell extracts were prepared by trichloroacetic acid precipitation with bead lysis (Keogh et al., 2006 (link)). These cell extracts were combined with loading buffer, boiled for 5 min at 95°C, and resolved via a 4–20% TGX SDS-PAGE gel (Bio-Rad). Proteins were transferred to a PVDF membrane, and then blocked with PBS Odyssey blocking buffer (Li-Cor, Inc.) for one hour with shaking at 22°C. The membrane was incubated overnight at 4°C while shaking in phosphate buffered saline +0.05% Tween-20 (PBST) containing 1:1000 dilution of anti-β actin antibody (mouse, ab8224, Abcam) and 1:500 dilution of an S. pombe-specific anti-Sir2p antibody (rabbit, generously provided by Dr. Allshire) (Buscaino et al., 2013 (link)). The membrane was washed once in PBST, and then agitated for 4 hr at 22°C with 1:10,000 dilution of goat anti-mouse IgG and 1:10,000 dilution of goat anti-rabbit IgG (IRDye 680RD and IRDye 800CW, respectively, Li-Cor, Inc.). The membrane was washed three times with PBST and imaged on an Odyssey CLx dual-channel imaging system (Li-Cor, Inc.).