Total protein was extracted by lysing cells in RIPA lysis buffer (KeyGene, Nanjing, China). After measurement of protein concentration, equal amounts of protein (30 μg per lane) were subjected to 10 % SDS-PAGE. After transferring onto the PVDF membranes (Bio-Rad), the membranes were blocked in 5 % skim milk (Beyotime), followed by immunoblotting with primary antibodies at 4℃for 10–14 h. Next, the corresponding secondary antibody (ab205718; 1:4000; Abcam, Cambridge, UK) was applied in combination with primary antibodies. Lastly, the combined signals were visualized with an enhanced chemiluminescence kit (KeyGene). In this study, the primary antibodies were purchased from Proteintech (Rosemont, IL, USA): E-cadherin (E-cad; 20874–1-AP; 2000), N-cadherin (N-cad; 22018–1-AP; 1:2000), GLUT1 (21829–1-AP; 1:2000), LDHA (21799–1-AP; 1:5000), ARF6 (20225–1-AP; 1:500) and β-actin (20536–1-AP; 1:2000).
Skim milk
Manufactured by Beyotime
Sourced in China, United Kingdom
Skim milk is a type of dairy product that has had the fat content removed, typically by centrifugation. It contains a high percentage of protein and low levels of fat and calories, compared to whole milk.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ab205718, by Abcam (4 mentions)
Ab181602, by Abcam (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Bio-Rad (2 mentions)
Enhanced chemiluminescence kit, by Keygene (2 mentions)
Ripa lysis buffer, by Keygene (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions)
Ab227387, by Abcam (2 mentions)
E cadherin e cad, by Proteintech (1 mentions)
N cadherin n cad, by Proteintech (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
β actin, by Proteintech (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Tead4, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
20 protocols using skim milk
1
Western Blot Analysis of Protein Markers
2
Western Blot Analysis of Protein Markers
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Cells were harvested and lysed with 1% NP-40 lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% NP-40, 1 mM PMSF, 50 mM NaF, 1mM Na3VO4 and 1X protease inhibitor cocktails (Sigma, P8340)). Proteins were separated in 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked with 5% skim milk (BD), incubated with primary antibodies (1:1000) in 5% skim milk at 4°C overnight, washed and incubated with HRP-conjugated secondary antibodies (Beyotime, 1:2000) in 5% skim milk for 1h at RT. The membrane was visualized by enhanced chemiluminescence reagents (Millipore). The following antibodies were used for Western blot: TEAD4 (1:1000; Abcam, ab58310), SIX1 (1:1000; Proteintech, 10,709-1-AP), E-cadherin (BD, 610,181), β-Actin (Sigma, A2228).
3
Western Blot Analysis of Protein Extraction
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Total protein was extracted with radioimmunoprecipitation assay buffer (Sigma, Saint Louis, Missouri) containing a protease inhibitor cocktail. After being quantified by a Pierce BCA Protein Assay Kit (Thermo, Waltham, Massachusetts), 20 µg protein was loaded into 4% to 12% Bis-Tris Gels (Thermo, Waltham, Massachusetts) followed by separation. Subsequently, the protein was transferred to polyvinylidene fluoride membrane (Millipore, Germany), and the membrane was blocked with 5% skim milk (Beyotime, China). Thereafter, the membrane was incubated with primary antibodies at 4°C overnight, followed by incubated with secondary antibody at 37°C. After incubation, the membrane was illuminated with NovexECL Chemiluminescent Substrate Reagent Kit (Invitrogen, Carlsbad, California). Finally, the images were taken and quantified by Image J (NIH, Carlsbad, California). Antibodies applied are shown in Table 2 .
4
Protein Analysis by Western Blotting
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Proteins were extracted with Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, China), and their concentrations were checked with BCA Protein Quantification Kit (Yeasen, China). After being separated using SDS-PAGE with PAGE Gel Quick Preparation Kit (12.5% and 10%) (Yeasen, China) and transferred onto the PVDF membrane (GE, USA), the unreacted sites were blocked with 5% skim milk (Beyotime, China) for 2 h and the primary antibodies against SLC7A5 (1 : 200, goat, ab99419), β-actin (1 : 500, goat, ab8229; 1 : 1000, rabbit, ab8227), p-mTOR (phospho-S2448, 1 : 1000, rabbit, ab109268), mTOR (1 : 1000, rabbit, ab32028), p-S6K1 (phospho-S424, 1 : 500, rabbit, ab131436), S6K1 (1 : 5000, rabbit, ab32529), p-4EBP (1 : 500, rabbit, ab47365), and 4EBP (1 : 2000, rabbit, ab32024) (Abcam, USA) were added and the samples were incubated at 4°C for 12 h. After being rinsed three times with TBST (Yeasen, China), the secondary HRP-conjugated donkey anti-goat antibody (1 : 1000, ab6885) or goat anti-rabbit antibody (1 : 2000, ab6721) (Abcam, USA) was administrated to the membrane and maintained for 1 h at room temperature. Next, the bands were washed with TBS-T and visualized using the 4CN HRP kit (Leagene, China). The relative expression levels were obtained via ImageJ 1.53f (NIH, USA).
5
Bone Tissue Protein Extraction and Western Blot Analysis
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Total protein was extracted from bone tissues using the bone tissue protein extraction kit (BestBio, China) following the manufacturer's instructions. Concentrations of proteins were measured using a BCA protein assay kit (Thermo Fisher Scientific, IL, USA). For western blot analysis, proteins were separated using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Subsequently, PVDF membranes carrying target peptides were blocked in 5% skim milk (Beyotime, China) and then incubated with primary antibodies against RANKL (1 : 1,000; PA5-110268, Invitrogen, CA, USA), OPG (1 : 1,000; ab73400, Abcam, UK), FoxO3a (1 : 1,000; ab23683, Abcam), Wnt1 (1 : 3,000; PA5-85217, Invitrogen), β-catenin (1 : 5,000; ab73400, Abcam), and GAPDH (1 : 10,000; ab181602, Abcam) at 4°C overnight. Next, membranes were incubated with secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1 : 2,000; ab6721, Abcam) for 2 h. Band intensity of protein peptide was detected using the enhanced chemiluminescence system with the ImagePro Plus software.
6
Experimental Lung Fibrosis Model
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The reagents included bleomycin (Nippon Kayaku Ltd., Tokyo, Japan), saline (Kelun Pharmaceutical Co., Sicuan, China), Pirfenidone (Beijing Continent Pharmaceuticals Co., Ltd, Beijing, China), Hydroxyproline assay kit instructions (#A030-2-1, Nanjing Jian Chen Bioengineering Institute, China), protease inhibitor (Roche Life Science, USA), BCA protein assay kit (BIO-RAD, USA), polyvinylidene fluoride (PVDF) membranes (Millipore, Germany), skim milk (Beyotime, China), chemiluminescence detection kit (SuperSignal™ West Pico Chemiluminescent Substrate, Thermo Scientific, USA), enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen, Austria), lysing solution (Merck, Germany). Primary antibodies used in our project were as follows: α-Smooth Muscle Actin (Cell Signaling Technology, #19245, USA), anti-collagen I (Cell Signaling Technology, #81375, USA), β-Actin or glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, #5174, USA), anti-CD3-PerCP (Invitrogen, Austria) anti-CD4-FITC (Biolegend, USA), anti-CD8-APC (Biolegend, USA). All horseradish peroxidase–conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
7
Protein Expression Analysis in Lung Tissue
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Homogenates of each lung tissue sample were lysed in a radio immunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Roche Life Science, USA) to extract the total protein. The protein concentration was determined using the BCA protein assay kit (BIO-RAD, USA). Equal amounts of total protein were gathered by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The membranes were incubated in a blocking solution containing 5% (w/v) skim milk (Beyotime, China) in TBST buffer and then incubated with primary antibodies overnight at 4 °C and the appropriate secondary antibodies at room temperature for 45 min. It was subsequently visualized with an enhanced chemiluminescence detection kit (SuperSignal™ West Pico Chemiluminescent Substrate, Thermo Scientific, USA). β-Actin or glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as the loading normal for the experimental data analysis.
8
Western Blot Analysis of CDK12 Protein
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To begin with, total proteins were obtained from tissues and cells via Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Sigma). Next, 40 μg proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 90 min, and instantly transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) for 1 h. Whereafter, PVDF membranes were blocked by 5% skim milk (Beyotime). After 3 h, primary antibodies anti-CDK12 (Abcam, ab37914, 1:1000) and anti-GAPDH (Abcam, ab181602, 1:3000) were incubated at the room temperature for 4 h. Then, goat anti-rabbit secondary antibody (Abcam, ab205718, 1:5000) was applied to conjugate with primary antibodies for 1 h. Finally, the immunoreactive signals were analyzed using the Image J software (NIH, Bethesda, MD, USA) after the detection of the enhanced chemiluminescence reagent (Sigma).
9
Western Blot Analysis of Apoptosis Markers
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Cells were lysed using RIPA lysis buffer (Beyotime) on ice and centrifuged (20 min, 14, 000g, 4°C) to extract total protein. Thereafter, the protein samples were denatured by heating at 100°C for 3–5 min. After measuring protein concentration with BCA protein assay kit (Abcam, Cambridge, UK), extracted protein samples (about 30 μg/lane) were separated using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Sangon Biotech), and protein was then transferred to nitrocellulose membranes (Invitrogen). After blocking with 5% skim milk (Beyotime), the membranes were then incubated with the primary antibodies at 4°C for 12–16 h: B-cell lymphoma-2 (Bcl-2; ab194583, 1:500, Abcam), BCL2-associated X protein (Bax; ab77566, 1:1000, Abcam), SOX4 (ab70598, 1:500, Abcam), β-actin (ab227387, 1:5000, Abcam). Afterwards, these membranes were probed with corresponding secondary antibody (ab205718/ab205719, 1:4000, Abcam). Finally, the combined signals were detected by enhanced chemiluminescence reagent (Solarbio, Beijing, China). Quantification of protein levels was determined using ImageJ software. The protein abundance was normalized by β-actin. All experiments were repeated three times.
10
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA buffer (Sigma, USA) and protein concentration was determined using BCA protein kit (Thermo, USA). 5 µg of each protein was added to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for electrophoresis. The proteins separated in the gel were transferred to a 0.22 µm diameter Immobilon-P membrane (Millipore, USA), and then the membrane was blocked in 5% skim milk (Beyotime, China) for 2 h at room temperature. Membranes were incubated with NCK1 (Abcam, USA), PD-L1 (Abcam, USA), GAPDH (Abways, China) antibodies overnight at 4 °C, and then added with Horseradish peroxidase-conjugated goat anti-rabbit/goat anti-mouse antibodies were incubated for 1 h at room temperature. Then ECL (Tanon, China) was used to detect the amount of protein.
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