To isolate proteins of fly heads (male and female), 500–1000 flies were frozen in liquid nitrogen and heads were separated with a sieve. The heads were pulverized with a sterile pestle and resuspended in buffer containing 10mM NaCl, 25 mM Hepes, pH 7.5, 2mM EDTA and 1x cOmplete™ Mini protease inhibitors (Merck). The suspension was kept on ice for 10 min with gentle mixing followed by centrifugation at 18300 x g at 4°C for 15 min. The pellet, containing larger membranous particles and the bulk of dSERT was resuspended in a buffer containing 10mM NaCl, 25 mM Hepes, pH 7.5, 1x cOmplete™ Mini protease inhibitors (Merck) and 0.5% CHAPS and incubated at 4°C for 10 minutes with gentle mixing. The solution was centrifuged at 3000 x g for 5 min to recover the membrane extract. 20 μg protein of each sample were used for western blotting according to standard procedures. The membrane was blocked in 5% milk and the primary rabbit anti-dSERT antibody (Giang et al., 2011) was used at a dilution of 1: 20 000 with a mouse anti-β-Actin (mAbcam 8224) at 1:10 000 used as a loading control.
Complete mini protease inhibitor
Manufactured by Merck Group
Sourced in United States
The Complete mini protease inhibitor is a laboratory product designed to inhibit the activity of proteases, which are enzymes that break down proteins. This product provides a comprehensive solution for the inhibition of a wide range of proteases, ensuring the preservation of protein samples during various research and analytical procedures.
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Lab products found in correlation
Odyssey blocking buffer tbs, by LI COR (3 mentions)
Mouse anti β actin, by Abcam (3 mentions)
Odyssey clx, by LI COR (3 mentions)
β actin, by Merck Group (2 mentions)
Goat anti rabbit 800, by LI COR (2 mentions)
Rabbit anti cypa, by Enzo Life Sciences (2 mentions)
Goat anti mouse 680, by LI COR (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Hdac1, by Abcam (1 mentions)
Hdac2, by Abcam (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Triton x, by Merck Group (1 mentions)
Np 40, by Merck Group (1 mentions)
X ray film, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
α sma, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
23 protocols using complete mini protease inhibitor
1
Isolating Drosophila Head Proteins
2
Cellular Fractionation for Protein Analysis
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Cell fractionation was performed using the protocol previously described by Me ´ndez and Stillman (2000) . Trypsinized cells were resuspended in medium containing serum, collected by centrifugation, and washed with ice-cold PBS. All procedures were performed on ice. Cells were resuspended into ice-cold Solution A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.34 M sucrose (Sigma-Aldrich), 10% glycerol, 1 mM dithiothreitol (Millipore), 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), and 1x Complete Mini protease inhibitors (Sigma-Aldrich). Triton X-100 (0.1%) (Sigma-Aldrich) was added, and the cells were incubated for 10 min at 4 C on a rotating wheel. Nuclei were collected in the pellet by low-speed centrifugation (4 min, 1,300 xg, 4 C). The supernatant was collected as the cytoplasm fraction and further clarified by high-speed centrifugation (15 min, 20,000 g, 4 C) to remove cell debris and insoluble aggregates. Nuclei were washed once in Solution A and then lysed in Solution B (3 mM EDTA, pH 8.0, 0.2 mM EGTA, 1 mM DTT, 1x Complete Mini protease inhibitors (Sigma-Aldrich)) by incubation for 30 min at 4 C on a rotating wheel. The lysate was further fractionated into nucleoplasm (supernatant) and chromatin (pellet) fraction by centrifugation at 1,700 g for 4 min at 4 C. The pellet was resuspended into Laemmli sample buffer and boiled at 98 C for 5 min before sonication.
3
Protein Expression Analysis of Cardiac Remodeling
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Scar and LV tissue were dissected from AMI, CHF, and sham hearts, respectively. Dissected tissues and isolated cells were homogenized in lysis buffer (50 mM Tris- HCl pH7.5, 150 mM NaCl, 0.5% NP-40 (Sigma), 0.5% Triton-X (Sigma), 1 mM EDTA (Sigma), and complete mini protease inhibitor (Roche). Protein concentrations were determined by BCA assay (Thermo Scientific). Typically, 40 μg of protein was loaded on 4% to 12% Tris-Bis gels (Life Technologies), separated in MOPS running buffer, and transferred to a PVDF membrane (Millipore). After blocking with 5% non-fat dry milk in 1× TBS, membranes were probed with HDAC1, HDAC2 (Abcam, 1:1,000), α-SMA (Sigma, 1:5,000), E-cadherin, GSK3β (Santa Cruz, 1:500), p-GSK3β, Cleaved Caspase 3, p-Akt, Akt (Cell Signaling, 1:1,000), β-Catenin active (Millipore,1:1,000), and β-Actin (Sigma, 1:50,000) in TBS with 5% milk overnight at 4°C. Following three washes in TBS, membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz, 1:50,000) for 1 h at room temperature. An ECL (Millipore) system was used for detection of the bands and exposed to X-ray film (Thermo Scientific) in a dark room. Densitometry analysis was performed with Alpha Ease FC software.
4
Protein Extraction and Western Blotting
5
VHH Anti-HA Immunoprecipitation and Immunoblotting
6
Cloning, Mutagenesis, and Purification of AlaRS Variants
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The E. coli alaS gene was cloned into the pET21b expression vector with a C-terminal His tag and purified from E. coli BL21(DE3) cells. Point mutations encoding amino acid substitutions (M217A, M658A, and M785A) were introduced into the alaS gene through PCR site-directed mutagenesis using two self-complementary oligonucleotides carrying the appropriate mutations (49 (link)). Following PCR mutagenesis, the reaction was treated with DpnI for 4 h at 37 °C to digest the template plasmid. The plasmids were then sequenced to confirm the introduction of the desired mutations.
The production of the AlaRS variants was induced at 37 °C for 4 h with 1 mM IPTG in LB media. The cells were spun down and resuspended in a buffer containing 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and EDTA-free cOmplete Mini Protease Inhibitor (Sigma). The cells were then lysed by sonication, clarified, and purified using a TALON metal affinity resin. AlaRS was eluted using 250 mM imidazole. The fractions containing the protein were concentrated using a 10 K Amicon concentrator and dialyzed into a final buffer containing 50 mM Tris–HCl (pH 7.5), 300 mM NaCl, and 50% glycerol (50 ).
The production of the AlaRS variants was induced at 37 °C for 4 h with 1 mM IPTG in LB media. The cells were spun down and resuspended in a buffer containing 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and EDTA-free cOmplete Mini Protease Inhibitor (Sigma). The cells were then lysed by sonication, clarified, and purified using a TALON metal affinity resin. AlaRS was eluted using 250 mM imidazole. The fractions containing the protein were concentrated using a 10 K Amicon concentrator and dialyzed into a final buffer containing 50 mM Tris–HCl (pH 7.5), 300 mM NaCl, and 50% glycerol (50 ).
7
Cytoskeleton Extraction of T. vaginalis
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As described above, the trophozoites were detached from the tube wall and washed twice in PBS (pH 7.2) with centrifugations at 1000× g for 5 min at room temperature (RT). For the cytoskeleton extraction, the washed cells were then resuspended in 1 mL of ice-cold PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2) containing 30% glycerol (Sigma–Aldrich, EUA), 2% Triton X-100 (Sigma–Aldrich, Saint Louis, MO, USA), 2% Igepal (Sigma–Aldrich), and 1 complete mini protease inhibitor (Sigma–Aldrich). The solution was vigorously vortexed at maximum speed for 2 min, incubated on ice for 2 min, and this was repeated two times. Then, the cytoskeleton-enriched fraction of T. vaginalis was washed in PBS (pH 7.2) with centrifugation at 17,000× g for 5 min at 4 °C. The success of the extraction was confirmed through light microscopy.
8
Protein Immunoblotting of O9-1 Cell Lysates
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Total protein lysates were generated from 80% confluent O9-1 cultures by lysis in PE-buffer (5 mol·L−1 urea, 10% glycerol, 0.1% SDS, 0.01 mol·L−1 Tris pH 6.8) with cOmplete Mini protease inhibitor (Sigma-Aldrich) and sonication. Protein concentrations were determined using the BioRad DC assay. After denaturation, 20 µg of protein were electrophoresed per lane alongside a molecular weight marker on SDS-polyacrylamide gels before blotting onto nitrocellulose membranes. Membranes were washed and blocked in 1× TBST with 5% bovine serum albumin before consecutive incubation with primary antibody (see Supplementary Table 3 ) and horseradish peroxidase-coupled protein A (Cymed) and detection using Clarity Western ECL Substrate (BioRad).
9
Protein Extraction and Western Blotting
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Cells were lysed in Hypotonic Lysis Buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, and cOmplete mini protease inhibitor (Sigma-Aldrich) for 20 min on ice. The lysates were mixed 1:1 with 2 × Laemmli buffer containing 1:20-diluted 2-mercaptoethanol, boiled for 10 min, and centrifuged at 16,000 × g for 5 min at 4°C. Samples were run on 4–20% SDS-PAGE and transferred to nitrocellulose membranes. Membrane blocking, as well as antibody binding were in TBS Odyssey Blocking Buffer (Li-Cor, Lincoln, NE). Primary antibodies used were rabbit anti-CypA (1:10,000 dilution; Enzo Life Sciences, Farmingdale, NY, catalogue #BML-SA296) and mouse anti-β-actin (1:1,000 dilution; Abcam, Cambridge, UK, catalogue #ab3280). Goat anti-mouse-680 (Li-Cor, catalogue #925–68070) and goat anti-rabbit-800 (Li-Cor, catalogue #925–32211) as secondary antibodies were used at 1:10,000 dilutions. Blots were scanned on the Li-Cor Odyssey CLx.
10
Western Blotting for p53 Quantification
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Cells were grown to 80% confluency in six-well plates (Greiner #5665-7160) and lysed using RIPA lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% IGEPAL CA-630 (Sigma #I9996), 0.1% sodium azide (Sigma #S8032), cOmplete mini protease inhibitor (Sigma #04693124001) or Halt protease and phosphatase inhibitor cocktail (Thermo Scientific #78442), 200 µM sodium fluoride (Sigma #S6776) and 200 µM of sodium orthovanadate (Sigma #450243). Cells were scraped, incubated on ice for 10 min and centrifuged at 7400 g. Lysates were quantified using the Pierce BCA kit (#23225). Samples were run on 4–20% TGX gels from BioRad (#567-1093) and blotted onto 0.45 µm PVDF membrane (GE Healthcare #10600023) using the BioRad semi-dry transfer system. Primary antibody for p53 protein from Sigma (#P6874) diluted to 1:200 and secondary HRP-linked anti-mouse antibody (Cell Signaling Technologies #7076) diluted to 1:3000 was used to visualize p53 bands. The p53 mutant proteins were seen using the V5 tag Ab (CST, Cat no 13202S). All blot images in each figure were from the same experiment and processed in parallel.
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