To test overexpression of candidate endonucleases, cells were washed in ice-cold 1× phosphate-buffered saline (PBS) and collected in ice-cold 1× PBS by scraping. The harvested cells were split into two portions: one for RNA isolation (see above) and one for protein isolation. Cells were pelleted by spinning at 2000 rpm for 5 min. The supernatant was removed and cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 2× protease inhibitor cocktail (Invitrogen)) for 30 min on ice. The lysate was cleared by centrifugation at 13 000 rpm for 10 min at 4°C. Protein lysate samples were electrophoresed through 4–12% Bis-Tris gels (Invitrogen). Following electrophoresis, protein lysate samples were transferred to a nitrocellulose membrane (GE Healthcare). The membrane was blocked in 5% milk in Tris-buffered saline tween (TBST). Washes and antibody dilutions were performed in TBST. The following antibodies and dilutions were used in this study: anti-FLAG M2 at 1:10 000 (Sigma) and anti-β-tubulin at 1:20 000 (Sigma). Membranes were imaged on an Amersham Imager 600.
Amersham imager 600
Manufactured by Cytiva
Sourced in United States, United Kingdom, China, Sweden
The Amersham Imager 600 is a versatile gel and blot imaging system designed for a wide range of applications. It offers high-quality image capture and analysis capabilities for various types of gels and membranes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (10 mentions)
Anti myc, by Abcam (9 mentions)
Anti ha mms 101r, by Fortrea (7 mentions)
Anti trim3 hap043396, by Merck Group (6 mentions)
Anti gfp, by Abcam (6 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ab9106, by Abcam (5 mentions)
Ab2302, by Abcam (5 mentions)
Anti gapdh, by Cell Signaling Technology (5 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Ab290, by Abcam (4 mentions)
Anti p53 sc 126, by Santa Cruz Biotechnology (4 mentions)
Anti actin gb12001, by Wuhan Servicebio Technology (4 mentions)
Mms 101r, by Fortrea (4 mentions)
Anti flag 20543 1 ap, by Proteintech (4 mentions)
Anti actin, by Merck Group (4 mentions)
Clarity western ecl substrate, by Bio-Rad (4 mentions)
170 protocols using amersham imager 600
1
Evaluating Endonuclease Overexpression in Cells
2
Detailed Western Blot Methodology
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Western blot were performed as previously described [13 (link)]. The following primary antibodies have been used: anti‐actin (Cell Signaling, Danvers, MA, USA, 4970), anti‐Drp1 (BD Bioscience 611113), anti‐pS616‐Drp1 (Cell Signaling 4494), anti‐pS637‐Drp1 (Cell Signaling 6319), anti‐Mfn2 (Abcam ab56889), anti‐Mfn1 (Santa Crux sc‐50330), anti‐Opa‐1 (BD Bioscience 612607), anti‐Fis1 (Abcam ab71498), anti‐Mff (Abcam ab129075), anti‐pT202/204‐ERK1/2 (Cell Signaling 4370), anti‐ERK1/2 (Cell Signaling 4695), anti‐Hsp90 (Cell Signaling 4877), anti‐pSer2481‐mTOR (Cell Signaling 2974), anti‐mTOR (Cell Signaling 2983), anti‐MnSOD (Enzo Life Sciences ADI‐SOD‐110), anti‐LC3B (Cell Signaling 3868), anti‐cFos (Cell Signaling 4384), anti‐GAPDH (Cell Signaling 2118), anti‐Akt1 (Cell Signaling 2938), and anti‐pThr308‐Akt (Cell Signaling 4056). All primary antibody incubations were followed by incubation with appropriated secondary HRP‐conjugated antibodies (GE Healthcare or Cell Signaling) in 5% milk plus 0.1% Tween‐20 (Sigma P2287). Detection of protein signals was performed using Clarity Western ECL substrate (Bio‐Rad 170‐5061) and Amersham Imager 600. Stripping of the membranes for reprobing has been performed using buffer containing 1% Tween‐20 (Sigma P2287), 0.1% SDS (Sigma 71729), and 0.2 M glycine (VWR M103) at pH 2.2 (two washes for 10 min).
3
Western Blot Analysis of Viral Proteins
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The proteins expressed in both systems were analyzed in a 12% SDS-PAGE gel followed by Western blot (Bio-Rad, Hercules, CA, USA). Western blotting analysis was performed using anti-GFLV CP polyclonal antibody (1:1000) (DSMZ, Braunschweig, Germany) and mouse anti-L2 polyclonal sera (kindly provided by Dr. Ebenezer Tumban, Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine). The development of the signals was carried out using the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) (Amersham™ Imager 600).
4
Western Blot Protein Detection
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Proteins are detected by Western blotting. After incubation, cell lysates were heated in 1x SDS loading buffer for 15 min at 100 °C. Proteins were separated by SDS-PAGE gel, after being transfered to 0.45 μm polyvinylidene fluoride membranes, blocked with 5% BSA for 1 h. Membranes were incubated with antibodies against cyclinB1, cyclinA2, CDC2, P21, PARP, p-JNK, and GAPDH at 4 °C overnight. Secondary antibody was incubated for 1 h. Bands were detected by a chemiluminescence imaging system (Amersham Imager 600) with Super ECL Detection Reagent (Yeasen, Shanghai, China). Results were analyzed by Image J.
5
Western Blot Detection of Virulence Factors
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After lysis, total protein concentration samples were measured via Bradford assay. Samples were subsequently diluted to 0.5 μg total protein/μl. All SDS-PAGE gels contained 12.5% acrylamide and were run at 90 to 120 V for 1.5 h. Proteins were transferred to nitrocellulose membranes using a semidry electroblotter (Fisher Scientific) overnight at 35 mA or for 2 h at 200 mA. Membranes were blocked with 5% nonfat milk, 2% bovine serum albumin in Tris-buffered saline, 0.5% Tween 20 (TBST) for 1 h. Membranes were then incubated with primary antibody (anti-TcpA, 1:100,000; anti-TcpP, 1:1,000; anti-TcpH, 1:500; anti-ToxR, 1:50,000; anti-mCherry, 1:1,000) diluted in TBST and nonfat milk (2.5%, wt/vol) for an additional hour at room temperature with shaking. Membranes were then washed 3 times with TBST. Secondary antibody (goat anti-rabbit IgG-horseradish peroxidase [HRP], 1:2,000) (Sigma) was diluted in TBST and nonfat milk (2.5%, wt/vol). Secondary antibody was incubated with the membranes for an additional hour at room temperature with shaking. Membranes were washed again with TBST 3 times and then incubated with SuperSignal HRP chemiluminescence substrate (Thermo Fisher). Membranes were imaged with an Amersham Imager 600.
6
Protein Expression Analysis of HNSCC Cells
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The total protein of HNSCC cells was extracted using SEMS lysis buffer (Beyotime, China); the protein contents were determined using a BCA protein assay kit (Beyotime, China). Equal amounts of proteins (40 μg) were separated using premade SDS-PAGE gels (Jinsirui, China) and transferred onto 0.22-μm polyvinylidene fluoride membranes (Merck Millipore, USA). The membranes were then incubated in 10% skim milk in PBS for 1 h at room temperature and incubated with the primary antibodies (1:1000) at 4°C overnight. The membranes were then probed with HRP-labeled rabbit antibodies (Beyotime, China) for 1 h, and an ECL (Beyotime, China) developing solution was added to enable the visualization of the protein bands, which were detected on a chemiluminometer (Amersham Imager 600). The antibodies against PLAU1, MMP1, MMP7, TIMP2, CDH1, and GAPDH were purchased from Abcam (UK).
7
Subcellular Fractionation and Immunoblotting
8
Western Blotting Protocol with Modifications
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Western blotting was performed as previously described15 (link) with modification. In brief, cells were lysed in buffer containing 20 mM Tris pH 7.5, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% NP-40, supplemented with 1:100 Halt protease and phosphatase inhibitor cocktail (Thermo Scientific) and benzonase (Novagen) at 12.5 U/mL. For lysis, cells were rotated at 4 °C for 30 min, supplemented with 1% SDS for boiling, and then the supernatants were subjected to electrophoresis and transfer to 0.2 μm nitrocellulose membrane. Membrane was incubated with primary antibodies in TBS supplemented with 0.1% Tween 20 at 4 °C for overnight, probed with HRP-conjugated secondary antibodies, and then was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and imaged by Amersham Imager 600.
9
Western Blot and Dot Blot Analysis
10
Western Blot Analysis of mTOR Pathway
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Three pairs ccRCC and adjacent tissues were chosen for WB analysis (Yuan et al., 2020 (link)). Membranes were incubated with the desired primary antibodies: mTOR (1:2,000, rabbit, ab32028; Abcam, Cambridge, MA, USA), p-mTOR (Ser2448, 1:2,000, rabbit, ab109268; Abcam, Cambridge, MA, USA), anti-Actin (1:1,000, mouse, BM0627; BOSTER, Beijing, China), the corresponding secondary antibodies (SA00001-2 & SA00001-1; Proteintech, Wuhan, China) were diluted to 1:5,000, and enhanced chemiluminescence (Amersham Imager 600; Marlborough, MA, USA) was used for immunodetection as previously described (Yuan et al., 2020 (link)).
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