After washing with pre-chilled sterile PBS, cells were subject to lysis through repeated freezing and thawing. After centrifugation at 12,000 rpm for 5 min, a BCA protein assay kit (Beyotime Ins. Biotech, China) was used to measure the protein content. Subsequently, the proteins were separated using 12% SDS-PAGE, followed by transfer onto nitrocellulose filter membrane (NC) membranes. Next, 5% bovine serum albumin (BSA) was added for a 2 h period to block membranes, followed by overnight primary antibody incubation (anti-TRAF3; Abcam; 1:1000，United Kingdom) under 4 °C. After rinsing with PBST, a secondary antibody (Goat Anti-Rabbit IgG H&L (HRP), Abcam; 1:50000，United Kingdom) was added and incubated for a 1 h under ambient temperature. Proteins of the membrane were visualized using enhanced chemiluminescence (ECL) reagent (Vazyme Biotech, China); autoradiograms were scanned and analyzed using Amersham ImageQuant 800 system (Cytiva，Japan). Calnexin was used as a loading protein.
Amersham imagequant 800 system
Manufactured by Cytiva
Sourced in Japan
The Amersham ImageQuant 800 system is a digital imaging platform designed for the analysis and quantification of biomolecules, including proteins, nucleic acids, and other biological samples. It utilizes high-resolution CCD imaging technology to capture and analyze images of stained gels, blots, and other samples.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti traf3, by Abcam (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Ecl reagent, by Vazyme (1 mentions)
Mouse anti flag m2 antibody, by Merck Group (1 mentions)
Sw40ti rotor, by Beckman Coulter (1 mentions)
Radioimmunoprecipitation assay buffer, by Bioss Antibodies (1 mentions)
Anti fto, by Abcam (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Anti gapdh, by Bioss Antibodies (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Goat anti rabbit, by Boster Bio (1 mentions)
Goat anti mouse, by Boster Bio (1 mentions)
Anti cyclind1, by Abways (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti c myc, by Abways (1 mentions)
Anti β actin, by Bioss Antibodies (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence solution, by Biosharp (1 mentions)
10 protocols using amersham imagequant 800 system
1
TRAF3 Protein Expression Analysis
2
Subcellular fractionation and immunoblotting
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Subcellular fractionation was performed as before (8 (link), 9 (link)). Briefly, cells were grown to a log phase (OD600, 0.5 to ~1.0), harvested, and lysed by nitrogen cavitation. The cell lysate was placed on top of a sucrose gradient (20 to 50%, wt/vol) and fractionated by sedimentation at 35,000 rpm for 6 h on an SW-40Ti rotor (Beckman-Coulter) at 4°C. SDS-PAGE and immunoblotting of gradient fractions were as previously described (8 (link), 45 (link)). Rabbit anti-PimB′ and anti-MptC antibodies were raised previously (63 (link)), affinity-purified, and used at 1.0 and 1.1 μg/mL, respectively. Mouse anti-FLAG M2 antibody was from Sigma-Aldrich and used at 1 μg/mL. Horseradish peroxidase-conjugated donkey anti-rabbit (Cytiva) or sheep anti-mouse antibody (Sigma-Aldrich) was used at a 4,000× dilution as a secondary antibody, and the protein bands were visualized by chemiluminescence. Ppm1-mNeonGreen was visualized by in-gel fluorescence imaging. Both luminescence and fluorescence were detected using either an ImageQuant LAS 4000mini or an Amersham ImageQuant 800 system (Cytiva).
3
Western Blot Analysis for Protein Expression
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Total proteins were extracted from the cells and tissues using radio immunoprecipitation assay buffer (BIOSS) mixed with phenylmethylsulfonyl fluoride (BIOSS) and quantified using bicinchoninic acid assay (Beyotime Institute of Biotechnology). Protein extractions (30ug per well) were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (MilliporeSigma). The membranes were blocked with 5% fat-free dried milk for 1 h at room temperature. After incubation with high-affinity anti-FTO (1:1000, Abcam, USA), anti-STAT3 (1:1000, Cell Signaling Technology, USA), anti- phosphorylation-STAT3 (1:1000, Cell Signaling Technology, USA), anti-Bcl-2 (1:1000, Cell Signaling Technology, USA), anti-c-Myc (1:1000, Abways Technology, China), anti-CyclinD-1 (1:1000, Abways Technology, China), anti-β-actin (1:5 000, Bioss, China), or anti-GAPDH (1:5000, Bioss, China) antibodies at 4°C overnight, the membranes were incubated with the HRP-conjugated secondary antibodies goat anti-rabbit (1:10,000; Boster, China) or goat anti-mouse (1:5000; Boster, China) for 1 h at room temperature. Proteins were detected using BeyoECL chemiluminescence kit (Biyuntian, China) and detected using the Amersham ImageQuant 800 system (Cytiva). The density of bands were measured using ImageJ (v1.51, National Institutes of Health).
4
Western Blot Analysis of Apoptosis Markers
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HeLa cells were lysed in 80 µL RIPA buffer (Beyotime, Shanghai, China) containing 0.8 µL 1 mM fluoride, and the protein concentration of each sample was determined using the BCA Protein Assay Kit (Bioss, Beijing, China). Proteins (20 µg) were boiled at 95 °C for 5 min, followed by separation by 15% SDS–PAGE. After electrophoresis, the proteins were electrophoretically transferred to PVDF membranes at 100 mA for 90 min. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CASP3, BCL−2, beta Actin (β−actin) (Proteintech Group, Inc., Chicago, IL, USA). The catalog numbers are 66470-2-Ig,60178-1-Ig, and 66009-1-Ig. Next, the membranes were treated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech Group, Inc., Chicago, IL, USA). The catalog number is SA00001-1. The membranes were visualized with enhanced chemiluminescence solution (Biosharp, Beijing, China) (A liquid:B liquid = 1:1) using an Amersham ImageQuant 800 system (Cytiva, Tokyo, Japan).
5
SARS-CoV-2 Receptor Expression Profiling
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Approximately 50mg of tissue samples were lysed in RIPA lysis buffer (Millipore, Massachusetts, United States) supplemented with protease inhibitor (Sigma Aldrich, St Louis, MO, United States) by TissueLyser II (Qiagen, Hilden, Germany). Total protein was collected and quantified using Pierce BCA protein assay kit (23225, Thermo Fisher Scientific, MA, United States). Protein samples were separated in NuPAGE Bis-Tris gel electrophoresis system (ThermoFisher Scientific, Massachusetts, United States) and transferred to PVDF membrane. The membranes were incubated with mouse anti-TMPRSS2 (1:1000, MA5-35756, Thermo Fisher Scientific, MA, United States), rabbit anti-ACE2 (1:1000, HPA000288, Atlas antibodies, Stockholm, Sweden), rabbit anti-NRP1 (1:1000, ab81321, abcam, Cambridge, United Kingdom), mouse anti-β actin (1:5000, ab8226, abcam, Cambridge, United Kingdom), respectively. The membranes were detected and imaged using Amersham ImageQuant 800 system (Cytiva, Uppsala, Sweden).
6
Western Blot Analysis of H1c Expression
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Twenty-four hours after transfection, radioimmunoprecipitation assay lysis buffer (Sangon Biotech, China) was added to the harvested cells to lyse the cells. The lysates were mixed with sample buffer and boiled for 10 min, after which the proteins in the sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Next, phosphate-buffered saline (PBS) with 0.1% Tween-20 (PBST) containing 5% nonfat dry milk powder was added to the membrane as a blocking solution. H1c expression was detected initially with anti-H1c rabbit polyclonal antibody (1:2000 dilution, in-house reagent) and then with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (1:5000 dilution, Biodragon, China). Later, the HRP substrate luminol reagent (Merck, Germany) was added to develop the exposure, and Amersham ImageQuant™ 800 system (Cytiva, Japan) was used for image capturing.
7
Labeling Protein Lysates with Bocillin-FL
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Cell lysates were prepared by bead beating as in the previous literature (Rahlwes et al., 2017 (link)). Then, 30 μg protein of cell lysate was incubated with 100 pmol Bocillin-FL (Invitrogen) in a total volume of 7.5 μL for 30 min in 37°C (Levine and Beatty, 2021 (link)). The sample was mixed with 2.5 μL of 4×-SDS-loading buffer and boiled for 3 min at 98°C. The entire sample was subjected to SDS-PAGE analysis. Gel was imaged by Amersham ImageQuant 800 system (Cytiva).
8
Protein Extraction and Western Blot Analysis
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The total proteins were extracted from the cells and mice tibia using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples each of 30 μg were separated by electrophoresis on a 10% sodium dodecyl sulfate–polyacrylamide gel (Bio-Rad Laboratories, Richmond, CA, USA) and transferred to 0.45 µm polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Little Chalfont, England). After blocking with 5% skim milk, the blots were incubated overnight at 4 °C with primary antibodies. All the membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. Finally, the western blots were imaged using an Amersham ImageQuant 800 system (Cytiva, Marlborough, MA, USA). The primary antibodies are listed in Supplementary Table 2 .
9
Western Blot Analysis of Mitochondrial Proteins
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Protein extracts from wt and CG1603 mutant larvae tissues (48h after egg laying) were prepared using the RIPA buffer (MilliporeSigma) with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), 5 mM NaF (MilliporeSigma) and 1 mM Na3VO4 (MilliporeSigma). Western blot was performed using a XCell SureLock™ Mini-Cell and XCell II™ Blot Module (Thermo Fisher Scientific). Samples were electrophoresed under a reducing condition on NuPAGE™ 4 to 12% Bis-Tris Mini Protein Gels (Thermo Fisher Scientific). Proteins on the gel were transferred to a Polyvinylidene Difluoride (PVDF) membrane (Thermo Fisher Scientific). The membranes were blocked with 5% BSA or non-fat milk (MilliporeSigma) in TBST (Tris buffered saline with 0.1% Tween-20). After a serial of washes and incubations with primary antibodies, TBST and secondary antibodies, the immunoreactivity was visualized using SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific) and Amersham ImageQuant 800 system (Cytiva). The antibodies used were: Mouse anti-Actin antibody (C4, MilliporeSigma), Mouse anti-ATP5A antibody (15H4C4, abcam), Mouse anti-ND30 antibody (17D95, abcam), rabbit anti-TFAM antibody (Liu et al., 2022 (link)), rabbit anti-HSP60 antibody (#4870, Cell Signaling), Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling) and Anti-mouse IgG, HRP-linked Antibody (#7076, Cell Signaling).
10
Western Blot Analysis of SOX9 Protein
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Cells were washed with PBS and scraped into RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.1% Na deoxycholate, 0.1% SDS in H2O with 1× protease inhibitor cocktail (Sigma-Aldrich 4693132001)), incubated on ice for 10 min, and sonicated to disrupt pelleted DNA using Bioruptor Plus (Diagenode). Sonicated lysates were incubated on ice for 10 min, and centrifuged at 16,000g for 10 min at 4 °C to pellet debris. Supernatants were normalized to the same protein content using the Pierce BCA Protein Assay kit (ThermoFisher, 23225), mixed with 4× SDS sample loading buffer (Invitrogen NP0007) and 0.1 M dithiothreitol (DTT), and boiled for 7 min. Samples were separated on Tris-glycine polyacrylamide gel electrophoresis (PAGE) gels in 1× Tris-glycine buffer with 0.1% SDS, transferred in 1× Tris-glycine buffer with 20% methanol, blocked in 5% milk + 1% BSA in PBST, immunoblotted with either SOX9 antibody (1:1,000, Sigma-Aldrich AB5535) or β-actin antibody (1:20,000, Abcam ab49900) overnight at 4 °C, probed with the appropriate secondary, developed using Pierce ECL Western Blotting Substrate (ThermoFisher, 32106), and imaged using an Amersham ImageQuant 800 system (Cytiva).
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