Rat chondrocytes were lysed in RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitor cocktail (Beyotime). The protein concentration was determined using the BCA Protein Assay Kit (Beyotime). Equal amounts of protein (50 µg) were separated by 10% SDS-PAGE (Beyotime) and transferred to PVDF membranes (Millipore). Following transfer, the membranes were blocked with 5% BSA in TBS containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies (COL 2, COL 10, SOX-9, and ACAN), directly diluted at a ratio of 1:1000 in 5% BSA-TBST. The following day, after washing thrice with TBST, the membranes were incubated with horseradish peroxidase-linked secondary antibodies, directly diluted at a ratio of 1:5000 (Abcam), for 1 h at room temperature. After washing three times with TBST, the membranes were visualized using the SuperSignal West Femto Kit (Thermo Fisher). Finally, the intensity of the blots was quantified using Image Lab 3.0 software (Bio-Rad). After washing three times with TBST, the membranes were visualized using the SuperSignal West Femto Kit (Thermo Fisher). Finally, the intensity of the blots was quantified using Image Lab 3.0 software (Bio-Rad) (Huang et al., 2017 (link)).
Image lab 3
Manufactured by Bio-Rad
Sourced in United States, Germany, China, United Kingdom
Image Lab 3.0 software is a comprehensive image analysis tool designed for the acquisition, analysis, and organization of gel and blot images. It provides intuitive tools for quantifying bands, spots, and other features in a variety of image file formats.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (42 mentions)
Chemidoc xr, by Bio-Rad (34 mentions)
Pvdf membrane, by Bio-Rad (23 mentions)
Electrochemiluminescence plus reagent, by Thermo Fisher Scientific (23 mentions)
Chemidoc xrs system, by Bio-Rad (22 mentions)
Gapdh, by Santa Cruz Biotechnology (17 mentions)
Pvdf membrane, by Merck Group (17 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (17 mentions)
Chemidoc xrs imaging system, by Bio-Rad (14 mentions)
Ripa lysis buffer, by Beyotime (13 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Ripa buffer, by Beyotime (11 mentions)
Bcl 2, by Cell Signaling Technology (10 mentions)
Polyvinylidene difluoride membrane, by Merck Group (10 mentions)
Nitrocellulose membrane, by Bio-Rad (9 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (9 mentions)
β actin, by Santa Cruz Biotechnology (8 mentions)
Gapdh, by Abcam (8 mentions)
Bca kit, by Beyotime (7 mentions)
Molecular imager chemidoc xrs, by Bio-Rad (6 mentions)
358 protocols using image lab 3
1
Chondrocyte Protein Expression Analysis
2
Western Blot Analysis of Phosphorylated AKT
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Frozen tissues were homogenized for Western Blot analysis. Protein concentration was equalized and proteins were separated with SDS-PAGE and transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Diegem, Belgium). Membranes were incubated overnight at 4°C with an antibody against pAKT (Ser473, Cell Signaling Technology, Leiden, Netherlands) or AKT (Cell Signaling Technology) in 5% bovine serum albumin. The following day, membranes were incubated with a secondary antibody containing horse-radish peroxidase (Goat-anti-rabbit: Bio-Rad, Veenendaal, Netherlands). To visualize the immune complex, membranes were treated with enhanced chemiluminescence reaction reagent and a picture was taken using Gel Doc XR+ Imaging system (Bio-Rad). Protein bands were analyzed using Image Lab 3.0.1 (Bio-Rad).
3
Western Blot Protein Detection Protocol
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Western blot analysis was performed as already described [32 (link), 40 (link)]. Briefly, for protein detection, primary antibodies anti-SGPL1 ((H-300) #sc-67368; Santa Cruz, USA), anti-SPHK1 ((M-209) #sc-48825; Santa Cruz, USA), anti-SPHK2 ((P-19) #sc-22704; Santa Cruz, USA), anti-PCNA ((PC10) #sc-56; Santa Cruz, USA), anti-β-Actin ((C4) #sc-47778; Santa Cruz, USA), anti-MTSS1 ((SS-3) #sc-101204; Santa Cruz, USA), anti-PARP1 ((B-10) #sc-74470; Santa Cruz, USA) and anti-Bcl-2 ((C-2) #sc-7382; Santa Cruz, USA) were incubated overnight at 4 °C followed by labeling with a horseradish peroxidase (HPR)-conjugated secondary antibody (mouse #7076; rabbit #7074P2; Cell Signaling, USA) for 1 h at room temperature. Finally, the protein signals were visualized with the Clarity™ Western ECL Chemiluminescent Substrate (Bio-Rad Laboratories Inc., USA). Stainfree-images and β-actin were used as loading control. Band intensity was analyzed densitometrically with the Molecular Imager ChemiDoc XRS and Image Lab 3.0.1 software (Bio-Rad, München, Germany). Protein detection was repeated at least three times with individually prepared cell lysates from independent passaged cells.
4
Protein Expression and Phosphorylation Analysis
5
Screening Cell Stress Proteins
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26 cell stress-related proteins (Table S1 ) were detected using their respective antibodies coated on the membrane from human cell stress array kit (R & D Systems, Minneapolis, MN, USA). For this, cells were treated with 0.05, 0.1 mM H2O2, and 2.5 mU/mL GOx for 24 and 48 h. The cell supernatants were then collected and stored at −80 °C for further cytokines analyses. Whereas cells were lysed for 30 min at 4 °C with a lysis buffer containing a cocktail of protease inhibitors (10 μg/mL Aprotinin, Leupeptin, and Pepstatin, respectively; all from R & D System, Minneapolis, MN, USA). The lysates were centrifuged at 14,000× g for 5 min at 4 °C. For each analysis, 250 µg of protein was pooled from the lysates of 5 individuals. Cell stress-related proteins were detected using the kit according to the manufacturer’s instructions. For this, chemiluminescence blots were recorded with ChemiDoc XRS (Bio-Rad Laboratories GmbH, Munich, Germany), and protein intensity was quantified by densitometric analysis of the blot using the Image Lab 3.0.1 software (Bio-Rad, Laboratories GmbH, Munich, Germany).
6
Western Blot Analysis of Phosphorylated ERK
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The cell lysates were diluted in Laemmli sample buffer with 2-mercaptoethanol and heated at 95 °C for 5 min. Samples (20 μg protein) were loaded on TGX-gels (Bio-rad) and run with Tris/glycine/SDS buffer at 200 V. After electrophoresis, the separated proteins were blotted to PVDF membranes using the Trans-Blot Turbo system with pre-packed transfer packs and the 3-min protocol (Bio-rad). After that, the blots were incubated in blocking buffer (Sigma Aldrich, Schnelldorf, Germany) at room temperature for 1 h. The primary antibody (r-α-human p-ERK; AF1018, R&D systems) was diluted in blocking buffer (0.2 μg/mL) and the membranes were incubated overnight at 4 °C. After washing, the blots were incubated with secondary antibody (0.5 μg/mL) and StrepTactin-HRP conjugate (1 μL/10 ml) for 1 h. After washing in PBS tween, the blots were added to a solution of luminol and peroxide buffer (Bio-rad) and the bands were detected by the ChemiDoc™ XRS+ system (Bio-rad) and analyzed with the software Image Lab™ 3.0.1 (Bio-rad).
7
Protein Extraction and Western Blot Analysis
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Whole cell lysates were prepared as previously described48 (link). Briefly, cells were treated with osimertinib, selumetinib, DNA-PK-I or AURK-A-I alone or in combination at the indicated concentrations for 72 h and then lysed with cold RIPA buffer (0.1% sodium SDS, 0,5% deoxycholate, 1% Nonidet, 100 mM NaCl, 10 mM Tris–HCl (pH 7.4), 0.5 mM DTT, and 0.5% PMSF) added with protease (Hoffmann-La Roche) and phosphatase (PhosSTOP; Roche Diagnostics) inhibitors, incubated 20 min on ice and clarified by centrifugation at 15,000 rpm for 20 min at 4 °C. Then, western blot analysis of proteins from different lysates was performed. Briefly, protein samples were resolved SDS-PAGE gel electrophoresis and transferred onto 0.2 µm nitrocellulose membranes (Trans-Blot Turbo; BioRad). After blocking membranes with selected antibodies, proteins were detected with Clarity Western ECL Substrate using the ChemiDoc (BioRad). Images were analyzed using BioRad software Image Lab 3.0.1.
8
Western Blot Protein Analysis
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Cell lysates were diluted in 2× Laemmli sample buffer with β-mercaptoethanol and boiled at 95 °C for 5 min. Samples (20 μg protein) were loaded on TGX-gels (any kD; Bio-rad, Sundbyberg, Sweden) and electrophoresed with Tris/glycine/SDS buffer at 200 V. The molecular weight standard was a Precision Plus Protein Western C standard. After electrophoresis, the separated proteins were blotted to a polyvinyl difluoride (PVDF) membrane using the Trans-Blot Turbo system with pre-packed transfer packs and the 3-min protocol (Bio-rad). The blots were incubated in blocking buffer (Sigma Aldrich, Schnelldorf, Germany) at room temperature for 1 h. The primary antibody was diluted in blocking buffer (1 μg/mL) and the incubation lasted for 1 h or overnight at 4 °C. Then, the blots were washed in PBS-tween 20 (5 × 10 min, 25 rpm). The blots were incubated with secondary antibody (1 μg/mL) and a StrepTactin-HRP conjugate (2 μL) for 1 h. A strenuous washing protocol was used (5 × 10 min, 40 rpm). Finally, the blots were added a solution of luminol and peroxide buffer (Bio-rad) and the bands were detected by the ChemiDoc™ XRS+ system (Bio-rad) and analyzed with the software Image Lab™ 3.0.1 (Bio-rad).
9
Western Blot Analysis of Protein Expression
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The cells were subjected to lysis using radioimmunoprecipitation assay (RIPA) buffer after incubation for 48 hours, and protein was quantified using a bicinchoninic assay (BCA) assay kit (Pierce Biotechnology/Thermo Fisher Scientific Inc.). Total proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes and blotted. The membranes were incubated with NKX2-1, EGFR, PI3K, p-AKT, and GAPDH at 4°C for 12 hours and washed with Tris-buffered saline-Tween (TBST) for 15 minutes. The membranes were incubated with rabbit anti-goat IgG for 1 hour at 37°C and washed with TBST for 15 minutes. The blots were visualized using enhanced chemiluminescence and analyzed using Image Lab 3.0 (Bio-Rad Laboratories Inc., Hercules, CA, USA).
10
Western Blot Analysis of Cellular Proteins
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Cellular nuclear protein was extracted using PIRA assay and the protein concentration was detected using a BCA kit (Beyotime Institute of Biotechnology, Haimen, China). 50 µg of protein were separated using 10% SDS-PAGE gel and transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in TBST for 2 h at room temperature, and incubated with primary antibodies MMP-28, TGF-β and GAPDH overnight at 4°C. Membranes were washed with TBST for 15 min and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime Institute of Biotechnology; 1:1,000 dilution) for 1 h at room temperature. Membranes were visualized using a Millipore Enhanced Chemiluminescence system and analyzed using Image Lab 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
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