Determination of contractile protein abundances per mg wet tissue were performed as previously described by our laboratory and others (Dowling et al., 2019 (link); Haun et al., 2019a (link)). Briefly, SDS-PAGE sample preps were made using 10 μl resuspended myofibrils, 65 μl distilled water (diH2O), and 25 μl 4× Laemmli buffer. Samples (5 μl) were then loaded on precast gradients (4–15%) SDS-polyacrylamide gels (Bio-Rad Laboratories; Hercules, CA, United States) and subjected to electrophoresis at 180 V for 40 min using pre-made 1× SDS-PAGE running buffer (VWR International; Randor, PA, United States). Following electrophoresis, gels were rinsed in diH2O for 15 min and immersed in Coomassie stain (LabSafe GEL Blue; G-Biosciences; St. Louis, MO, United States) for 2 h. Gels were then destained in diH2O for 60 min, and band densitometry was performed using a gel documentation system and associated software (ChemiDoc; Bio-Rad Laboratories). Given that a standardized volume from all samples was loaded onto gels, MHC and actin band densities were normalized to wet muscle weight to derive relative protein abundances expressed as arbitrary density units (ADU) per mg wet muscle. Our laboratory has reported that this method yields good sensitivity in detecting 5–25% increases in relative MHC and actin protein abundances (Roberts et al., 2018 (link)).
Sds polyacrylamide gel
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SDS-polyacrylamide gel is a laboratory equipment used for the separation and analysis of proteins based on their molecular weight. It uses a technique called sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins under denaturing conditions.
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Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (30 mentions)
Pvdf membrane, by Merck Group (27 mentions)
Bradford assay, by Bio-Rad (16 mentions)
Pvdf membrane, by Bio-Rad (16 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Bovine serum albumin (bsa), by Merck Group (12 mentions)
Ripa buffer, by Merck Group (12 mentions)
Clarity western ecl substrate, by Bio-Rad (11 mentions)
Cell lysis buffer, by Cell Signaling Technology (10 mentions)
Protease inhibitor cocktail, by Merck Group (10 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (9 mentions)
Nitrocellulose membrane, by GE Healthcare (9 mentions)
β actin, by Abcam (8 mentions)
Dc protein assay, by Bio-Rad (8 mentions)
Image lab software, by Bio-Rad (8 mentions)
Anti β actin, by Merck Group (8 mentions)
Ripa buffer, by Cell Signaling Technology (8 mentions)
Chemidoc xrs system, by Bio-Rad (7 mentions)
β actin, by Merck Group (7 mentions)
334 protocols using sds polyacrylamide gel
1
Quantifying Contractile Protein Abundances
2
Western Blot Analysis of Oxidative Stress Markers
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Total cell lysate proteins were extracted and collected after the treatments. The protein concentration was detected by using the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). After the determination of total protein concentrations, the samples were denatured and loaded (40 μg/well) onto sodium dodecyl sulfate (SDS) polyacrylamide gels (Bio-Rad, Hercules, CA, USA) for electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF) (Bio-Rad, Hercules, CA, USA) membrane by using electrophoretic transfer (Bio-Rad, Richmond, USA). Nonspecific reactivity was blocked for 1 h at room temperature with standard 1X tris-buffered saline—Tween 20 (TBST) (Sigma-Aldrich, St. Louis, MO, USA) buffer containing 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) and 0.02% sodium azide. Primary antibodies of superoxide dismutase 1 (SOD-1), catalase, p53, Bad, Bax, Bcl-2, cytochrome c and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were probed and visualized on Li-COR C-digit Blot scanner (LI-COR Biosciences, Lincoln, Nebraska, USA) by using chemiluminescent reagent containing luminol.
3
Western Blot Analysis of Protein Signaling
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Samples were denatured in buffer containing 60 mM Tris/pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate (SDS), and 14.4 mM 2-mercaptoethanol with 0.1% bromophenol blue, and boiled for 5 minutes. SDS polyacrylamide gels (Bio-Rad, Hercules, CA, USA) were loaded with 25 µg of total protein per lane. Prestained molecular weight markers (Bio-Rad) were used as standards. Electrophoresed samples were transferred to polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ, USA). After transfer, membranes were blocked with 3% bovine serum albumin in Tris-phosphate-buffered saline (TPBS; 200 mM Tris/pH 7.0, 1.37 M NaCl, 1% Tween-20) for 1 hour at room temperature. Membranes were then incubated with anti-RET, anti-phospho-RET, anti-EGFR, anti-phospho-EGFR, anti-AKT, anti-phospho-AKT, anti-ERK, anti-phospho-ERK, or anti-beta actin (1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4℃, washed as before, and incubated with goat anti-rabbit peroxidase-conjugated secondary antibody (1:4000; Cell Signaling Technology) for 2 hours at room temperature. Membranes were then washed, and expressed proteins were detected with Pierce ECL plus western blotting substrate (Pierce, Rockford, IL, USA). Experiments were conducted at least three times independently.
4
Immunoblotting Analysis of Intracellular Signaling
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Cancer cells and BMFs were lysed with lysis buffer. Protein samples were subjected to SDS-polyacrylamide gels (Bio-Rad) electrophoresis. The gels were transferred onto nitrocellulose membranes (Bio-Rad). The membranes were probed with specific primary antibodies against JAK2 (Cat. 3230S), p-JAK2 (Cat. 3771S), STAT3 (Cat. 9139S), p-STAT3-Y705 (Cat. 76315), Met (Cat. 8198), p-Met (Cat. 3077), Smad2/3 (Cat. 5678), p-Smad2 (Cat.8828), vimentin (Cat. 3879) (Cell signaling Technology), snail (Cat. 92547), E-cadherin (Cat. ab15148), TGF-β1 (Cat. ab66043 ), and IL-6 (Cat. ab6672), and β-actin (Cat. ab6276) (Abcam), incubated with secondary antibodies conjugated to IR fluorophore, Alexa Fluor 680 (Molecular Probes), or IRdye 800 (Rockland Immunochemicals). Antigen-antibody complexes were visualized by the ECL system (Amersham Biosciences, Piscataway, NJ) and scanned using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The experiments were repreated three times (n=3).
5
Western Blot Analysis of UPR Markers
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Caco-2 monolayers were lysed directly in Laemmli buffer supplemented with 5% (v/v) 2-mercaptoethanol and boiled for 15 minutes. Equal amounts of lysate were separated by electrophoresis on SDS-polyacrylamide gels (Biorad) under denaturing conditions. Proteins were transferred to PVDF membranes (Millipore) via wet electrotransfer. After protein blotting, membranes were blocked in PBS with 0.1% (v/v) Tween-20 with either 5% (w/v) non-fat dry milk or 5% (w/v) BSA. Primary antibodies against active Caspase-3 (BD Biosciences), CHOP (Cell Signalling), GRP78 (Cell Signalling), XBP1s (BioLegend), or βActin (Santa Cruz) were applied overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies raised against mouse, rabbit or goat immunoglobulins (Abcam). Protein bands were visualized with ECL (Pierce) and ChemiDoc MP System (Bio-Rad). Quantification of the bands were performed by imageJ software, with the intensity values normalized to the corresponding βActin band.
6
L. pneumophila Infection in BMDMs
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BMDMs were plated at 1.56× 105 cells per cm2 on Costar Clear Not Treated 6-well plates with RPMI containing 10% L-cell supernatant/10% FBS. After 24 h, cells were challenged with L. pneumophila at MOI of 15, followed by contacting the BMDMs in the centrifuge at 400 RCF for 10 min. At the indicated time points, the medium was removed and adherent cells were lifted with ice-cold PBS and pelleted at 1,000 RCF for 5 min in the centrifuge. Cells were sorted and lysed with 1 × SDS Laemmli sample buffer (0.125 M Tris-Cl pH 6.8, 4% SDS, 20% glycerol, 10% beta-mercaptoethanol, 0.01% bromophenol blue). Cell lysates were boiled for 5 min and fractioned on SDS-polyacrylamide gels (Bio-Rad) and then electroblotted on nitrocellulose membranes. Blots were blocked with 5% BSA in Tris-buffered saline-tween (TBST: 0.05 M Tris-HCl pH 8.0, 0.138 M NaCl, 0.0027 M KCl, 0.05% Tween 20). For immune detection, cells were probed with a primary antibody (1:1,000 or manufacturer’s recommendation) in 5% BSA/TBST overnight at 4°C. After being washed with TBST, secondary antibodies Dylight anti-rabbit IgG 680, Dylight anti-mouse IgG 680, Dylight anti-rabbit IgG 800, or Dylight anti-mouse IgG 800 (Cell Signaling 1:20,000) were incubated in 4% milk/TBST for 45 min at room temperature. The membranes were scanned by Odyssey Imaging System and the Image Studio software (LI-COR Biosciences).
7
Platelet Protein Synthesis Assay
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Leukocyte- and erythrocyte-depleted washed PLTs were incubated in complete DMEM/Tyrode’s albumin medium (1/1 v/v) at 37°C for various periods of time before sampling (after 10 min, 5 h 30 and 23 h 30). PLT samples were washed using two serial centrifugations at 1900g in cysteine-free DMEM/Tyrode’s albumin buffer supplemented with 0.5 μM PGI2, incubated in methionine- and cysteine-free DMEM/Tyrode’s albumin buffer for 30 min at 37°C, washed again using two serial centrifugations in cysteine-free DMEM/Tyrode’s albumin buffer supplemented with 0.5 μM PGI2 and then labeled in methionine- and cysteine-free DMEM/Tyrode’s albumin medium supplemented with 35S methionine and cysteine (0.5 mCi/109 PLTs/1 mL, Perkin-Elmer) for 30 min at 37°C After labeling, the PLTs were washed using two serial centrifugations in methionine- and cysteine-free DMEM supplemented with 0.5 μM PGI2 and platelet pellet was lysed in 100 μL of 2% SDS at 95°C for 5 min. Protein concentrations were determined using a BCA assay (Thermo Scientific) and 25 μg samples were loaded onto SDS polyacrylamide gels (4–15%, Biorad). After electrophoresis, the gels were stained with Coomassie blue G250, fixed, dried and autoradiographed (Amersham Hyperfilm MP, D Dutscher).
8
Chromatin-Bound Protein Analysis Protocol
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For analysis of chromatin-bound proteins, we used a protocol slightly modified from Räschle et al., 2008 (link). Briefly, reactions were diluted into a 13-fold volume of ELB buffer (10 mM Hepes pH 7.5, 50 mM KCl, 2.5 mM MgCl2) containing 1 mM DTT, 0.2% Triton X100, protease inhibitors and phosphatase inhibitors. Chromatin was recovered through a 500 mM sucrose cushion in ELB buffer at 6780 g for 50 s at 4 °C, washed twice with 200 µl of 250 mM sucrose in ELB buffer, and resuspended in 20 µl SDS sample buffer. Western blots were conducted using standard procedures on Xenopus embryo/tadpole protein extracts. Proteins were loaded, separated by 7.5%, 12%, or 4–15% SDS-polyacrylamide gels (Bio-Rad) and transferred into nitrocellulose or ImmobilonP membranes. Membranes were subsequently incubated with the indicated primary antibodies followed by the appropriate horseradish peroxidase-labelled antibodies (1/10,000, Sigma-Aldrich or GE Healthcare, see Key Resources Table). Immunodetection was performed using Super Signal West Pico or Femto Chemiluminescence Kit (Pierce). Quantification was done using Fiji software (Schindelin et al., 2012 (link)) or using Biorad ImageLab software.
9
Western Blot Analysis of ACE2, p38, and NF-κB
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Total proteins were extracted using RIPA buffer (Euromedex, Souffelweyersheim, France), and then equal amounts of proteins were reduced, size-separated on 12% stain-free precast SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA), and transferred to nitrocellulose membranes by using an iBlot2 apparatus (Thermo Fisher Scientific). The membranes were blocked in 5% milk in TBS-Tween 0.1% and incubated with specific primary antibodies overnight at 4°C; the antibodies were against ACE2 (AF933, R&D Systems, Minneapolis, MN, USA; 1:200), phospho- and total p38 (9211 and 9212, Cell Signaling Technology, Danvers, MA, USA; 1:2,000), phospho- and total NF-κB p65 (3039 and 8242, Cell Signaling Technology; 1:2,000), and β-actin (A2228, Sigma-Aldrich; 1:5,000). The blots were exposed to horseradish peroxidase-conjugated anti-rabbit (Cell Signaling Technology, 7074; 1:10,000) and anti-goat (A27104, Thermo Fisher Scientific; 1:2,000) secondary antibodies, and bound antibodies were detected using Clarity chemiluminescent substrate (Bio-Rad). Images were recorded using a Fujifilm LAS-3000 bioimaging system (Stamford, CT, USA).
10
Western Blot Analysis of Francisella Proteins
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Unless stated otherwise, lysates from bacteria grown on modified GC agar were prepared in Laemmli sample buffer and boiled prior to separation on 12–15% sodium dodecyl sulfate (SDS)-polyacrylamide gels (161–0175, Bio-Rad). Proteins were transferred onto nitrocellulose membranes (170–4158, Bio-Rad) using a semidry blotter (Bio-Rad laboratories, Hercules, CA). Membranes were probed with mouse monoclonal antibodies recognizing IglC or PdpB at 1/2,000 and 1/1,000 dilutions respectively (NR-3196 and NR-3198, BEI Resources), rabbit polyclonal antibodies recognizing VgrG at 1/1,000 dilution (Inbiolabs, Tallinn, Estonia), mouse monoclonal antibodies recognizing Tul4 (1/3,000 dilution) (kind gift from Dr J. Stulik, Czech Republic), rat polyclonal antibodies recognizing IglE 34 (link) (1/2,000 dilution). GSK-tagged variants of IglE were detected using rabbit anti-GSK antibodies (1/1,000 dilution) (9336S, Cell Signaling Technology). The secondary horseradish peroxidase (HRP)-conjugated antibodies used were: goat anti-mouse and goat anti-rat (S-2005 and S-2032, Santa Cruz Biotechnology) at 1/2,000 and 1/5,000 dilutions respectively, and donkey anti-rabbit (NA934V, GE Healthcare) at 1/13,300 dilution. For detection, the Enhanced Chemiluminescence system (ECL; RPN 2232, GE Healthcare) was used.
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