Cells were seeded on 10-cm-diameter dishes until 80-90% confluency was attained. On the day of the experiment, cells were rinsed once with 5 mL of serum free Opti-MEM and then stimulated with 1 μM LPA prepared in the chemotaxis assay buffer (0.1%BSA in RPMI1640) pre-warmed at 37 °C for 30 s, 2 min, or 5 min. Immediately after stimulation, the medium was replaced with ice-cold chemotaxis assay buffer and cells were kept on ice until lysis was done. Cells were lysed with ice-cold lysis buffer from the PathScan RTK Signaling Antibody Array kit (Cell Signaling Technology, Danvers, MA, USA) per the manufacture’s procedure. Cell lysate was kept at −70 °C until the PathScan phosphorylation array or SDS-PAGE/western blotting was performed. For western blotting, each cell lysate was subjected to SDS-PAGE, blotting, and antibody reaction. The pre-stained protein marker (Bio-Rad, Hercules, CA, USA) or the CruzMarker protein marker (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to estimate the molecular weight of probed bands. Protein bands were visualized with ECL prime (GE Healthcare) and detected by LAS-4000 mini device (GE Healthcare). The list of the phosphorylated proteins for the array is shown in Additional file 2 : Table S2.
Pre stained protein marker
Manufactured by Bio-Rad
Sourced in United States
The Pre-stained Protein Marker is a molecular weight standard used to estimate the size of proteins in SDS-PAGE and Western blot analysis. It consists of a mixture of pre-stained proteins with defined molecular weights, which are visualized by a built-in dye.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence kit, by Bio-Rad (2 mentions)
Tris glycine, by Bio-Rad (2 mentions)
Tetramethylethylenediamine, by Bio-Rad (2 mentions)
Hrp conjugated goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
Protease phosphatase inhibitor mixture, by Applygen (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pathscan rtk signaling antibody array kit, by Cell Signaling Technology (1 mentions)
Las 4000 mini device, by GE Healthcare (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Sodium dodecyl sulphate (sds), by Merck Group (1 mentions)
Endothelial cell growth supplement (ecgs), by Merck Group (1 mentions)
M199 medium, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
9 protocols using pre stained protein marker
1
LPA-Induced RTK Signaling Pathway
2
Puerarin Endothelial Cell Signaling Protocol
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Puerarin was purchased from the Beijing Union Pharmaceutical Factory (Beijing, China). M199 medium was from Gibco Co. (Carlsbad, CA, USA). Endothelial cell growth supplement (ECGS), collagenase I, tetramethyl ethylene diamine (TEMED), phenylmethyl sulfonylfluoride (PMSF), sodium dodecyl sulphate (SDS), β-glycerophosphate, sodium orthovanadate, leupeptin, DTT, and anti-Factor VIII antibody were from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies for total and phospho-p42/p44-MAPKs were from Santa Cruz Biotechnology. PD98059 was from Calbiochem. Prestained protein marker was from Bio-Rad Co. Antibodies specific for nonphosphorylated and phospho-specific p42/p44-MAPKs and avidin-biotin-peroxides complex (ABC) kit for immunohistochemistry were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Low-molecular-weight calibration kit for SDS electrophoresis was from Amersham Biosciences Co. (Buckinghamshire, UK); cocktail tablets were from Roche Co. (Basel, Switzerland).
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Puerarin was purchased from the Beijing Union Pharmaceutical Factory (Beijing, China). M199 medium was from Gibco Co. (Carlsbad, CA, USA). Endothelial cell growth supplement (ECGS), collagenase I, tetramethyl ethylene diamine (TEMED), phenylmethyl sulfonylfluoride (PMSF), sodium dodecyl sulphate (SDS), β-glycerophosphate, sodium orthovanadate, leupeptin, DTT, and anti-Factor VIII antibody were from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies for total and phospho-p42/p44-MAPKs were from Santa Cruz Biotechnology. PD98059 was from Calbiochem. Prestained protein marker was from Bio-Rad Co. Antibodies specific for nonphosphorylated and phospho-specific p42/p44-MAPKs and avidin-biotin-peroxides complex (ABC) kit for immunohistochemistry were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Low-molecular-weight calibration kit for SDS electrophoresis was from Amersham Biosciences Co. (Buckinghamshire, UK); cocktail tablets were from Roche Co. (Basel, Switzerland).
3
Quantifying XTH/XET Protein Levels
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To determine XTH/XET protein levels, 20 μg of cell wall proteins were separated in 12% sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis. Anti-XTH/XET (Agrisera, Vännäs, Sweden) were used as primary antibodies (diluted 1/500) and anti-mouse antibodies (Bio-Rad, Hercules, CA, United States) were used as the secondary antibodies. Immuno-blotting was performed according to the standard protocol. Bands for XTH/XET located at 33 kDa were determined on the basis of a pre-stained protein marker (Bio-Rad) as the reference. The relative protein levels were quantified by densitometry analysis using Image-Lab 5.2. software (Bio-Rad).
4
MALDI-TOF/TOF MS Analysis of RP-HPLC Protein Fractions
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Active RP-HPLC protein fractions were subjected to SDS-PAGE using Laemmli method [21 ], prior to MALDI-TOF/TOF MS analysis. The protein separation was done on 16% polyacrylamide Tris/HCl gels. Prestained Protein Marker, broad range 6–175 kDa (Bio-Rad), was used as a molecular weight marker. Samples were boiled at 90°C for 5 minutes prior to electrophoresis. The electrophoresis was carried out using Bio-Rad Power Pac 300 at a constant voltage of 60 V for the first 15 minutes, followed by gradual increase to 80–100 V. The gels were then stained with Coomassie Brilliant Blue R-250 and documented. In order to improve visualisation of faint protein bands, MS compatible silver staining method was employed. The gels' images were captured for identifications. Excised protein bands from silver-stained SDS-PAGE gel were subjected to in-gel tryptic digestion and later prepared for MALDI-TOF/TOF MS analysis. Intact RP-HPLC protein fractions were also subjected to in-solution tryptic digestion and MALDI-TOF/TOF MS.
5
Molecular Mechanisms of Anti-cancer Drug Action
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The antibodies against P-STAT3, STAT3, and GAPDH were purchased from Cell Signal Technology (Danvers, MA, USA). The horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The caspase-3 colorimetric assay kit was purchased from Abcam Co. (Cambridge, MA, USA). A Bradford protein-assay kit, polyvinylidene fluoride membrane, and enhanced chemiluminescence kit were obtained from Bio-Rad Laboratories (Hercules, CA, USA). A protease phosphatase-inhibitor mixture was obtained from Applygen Technologies (Beijing, People’s Republic of China). Acrylamide (30%), coomassie brilliant blue, tetramethylethylenediamine, tris-glycine, sodium dodecyl sulfate, prestained protein marker, and nonfat dry milk were from Bio-Rad Laboratories.
6
SDS-PAGE Analysis of Peptide MW
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Molecular weight distribution of peptides in the collected supernatants was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) following Laemli’s protocol [33 (link)]. Aliquoted supernatant samples were treated using a loading buffer containing 10% (w/v) SDS, 50% (v/v) glycerol, 500 mM DTT, 0.25 M Tris-HCl, pH 6.8, and 0.05% (w/v) bromophenol blue and subsequently loaded into the gel wells. Peptide separation was performed at a constant voltage of 120 V on 7% or 12% acrylamide gels. Pre-stained protein marker (Bio-Rad Laboratories, Hercules, CA, USA) was used as the molecular weight standard.
7
Detecting CSGA-GFP Fusion Protein in P. pastoris
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Presence of E. coli CSGA-GFP protein in P. pastoris was confirmed by detecting CSGA-GFP fusion protein using polyclonal rabbit anti-GFP antibodies (from Life Technologies USA; cat # A-11122). Amount of protein was normalized, based on either the number of cells or by dry mass of cells. Cells were treated, with 12.5 % TCA and samples were incubated at -80 °C for one hour or overnight. On completion of incubation, samples were thawed, at room temperature and, the samples were centrifuged, at 12000 g for 8 min. The supernatant was, discarded and, the pellet was, washed twice with 80 % chilled acetone. Finally, protein pellet was air-dried and was resuspended, in 150 µL 1 % SDS and 0.2 N NaOH. Then 150 µL 2X dye (100 mM Tris HCl pH6.8, 200 mM DTT, 4 % SDS, 0.2 % bromophenol blue, 20 % glycerol) was added to the sample and samples were heated for 5 min at 95 °C using dry heating block. Samples were cooled down, to room temperature, vortexed, spanned and, an equal amount or volume of samples was loaded, in each well of 10 % SDS-PAGE along with pre-stained protein marker (Biorad, cat # 161-0376). Samples were run at constant 100V till the dye front reaches the bottom of the gel (Kumar 2019).
8
Ginsenoside Rh2 Modulates Doxorubicin-Induced Toxicity
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Doxorubicin hydrochloride (Dox), Retinoic acid (RA), Xylene, Ethanol, Tween‐20, and Methanol were purchased from Sigma‐Aldrich (St. Louis, MO. USA). Difco™ Skim Milk was purchased from BD Company (Franklin Lakes, NJ, USA). Fetal bovine serum (FBS), Penicillin, streptomycin, were purchased from Gibco‐Invitrogen (Grand Island, NY), Laemmli buffer, polyvinylidene difluoride (PVDF) membranes, nitrocellulose membranes, Proceau solution, Tris‐glycine buffer, Tris‐glycine‐SDS buffer, pre‐stained protein marker were purchased from Bio‐Rad (Hercules, CA, USA). Live and fixed cell nuclear imaging kit, Novex 4% to 12% tris‐glycine gels, Bolt™ 12%, Bis‐Tris gels, iBlotTM Transfer Stack, PVDF were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Masson trichrome (MT) and Sirius Red (SR) staining kits were obtained from Abcam Company (Cambridge, UK). Ginsenoside Rh2 with a purity of more than 98% was prepared with HPLC by our research group.
9
Gracillin Inhibits STAT3 Signaling in Vitro
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Gracillin (HPLC > 98%) was purchased from Herbest (Shanxi, China). The compound used in vitro was dissolved in dimethyl sulphoxide (DMSO). The antibodies against STAT3, STAT1, P‐STAT5, STAT5, P‐STAT4, STAT4 and GAPDH were purchased from Cell Signaling Technology. The antibodies against P‐STAT3, Survivin, VEGF, Mcl‐1, BAX, cleaved caspase3, P‐STAT1 and lamin‐B were purchased from Abcam Co. The horseradish peroxidase (HRP)‐conjugated donkey anti‐rabbit IgG, HRP‐conjugated goat anti‐mouse IgG and the anti‐Bcl‐2 antibody were purchased from Santa Cruz Biotechnology Inc. The Annexin V‐FITC Apoptosis Detection Kit I was obtained from BD Pharmingen. Methylthiazolyldiphenyl‐tetrazolium bromide (MTT) and DMSO were obtained from Sigma‐Aldrich Co. Hoechst 33 258 was purchased from Beyotime Institute of Biotechnology. The Lipofectamine 3000 was purchased from Thermo Fisher Scientific. siRNA was purchased from GenePharma. The Dual‐Luciferase Report Assay Kit was obtained from Promega Biotech Co., Ltd. A Bradford protein‐assay kit, polyvinylidene fluoride membrane, enhanced chemiluminescence kit, acrylamide (30%), Coomassie Brilliant Blue, tetramethylethylenediamine, Tris‐glycine, sodium dodecyl sulphate, pre‐stained protein marker and non‐fat dry milk were obtained from Bio‐Rad Laboratories. The protease phosphatase‐inhibitor mixture was obtained from Applygen Technologies.
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