Precooled RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) was used to extract total protein from cells and tissues. The BCA Protein Assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology) was used to determine protein concentrations. Proteins (60 µg per lane) were separated on 10% SDS-PAGE. Subsequently, the proteins were transferred onto polyvinylidene fluoride membranes (cat. no. 32031602; MilliporeSigma). Then, the membranes were blocked with 5% fat-free milk at room temperature for 2 h. Following blocking, the membranes were incubated with primary antibodies (1:1,000, anti-FANCI, cat. no. ab74332; 1:1,000, anti-FANCD2, cat. no. ab108928; cat. no. Abcam) 1:1,000, anti-UBE2T, cat. no. ab179802; 1:1,000, anti-WDR48, cat. no. ab230645; 1:1,000, anti-E-cadherin, cat. no. ab40772; 1:1,000, anti-N-cadherin, cat. no. ab76011; and 1:1,000, anti-Vimentin, cat. no. ab92547) purchased from Abcam at 4°C overnight, followed by incubation with corresponding secondary antibodies for 1 h at room temperature. β-actin (1:2,000, cat. no. ab8226; Abcam) was used as an internal reference protein. The membranes were visualized using ECL solution (E411-04, Vazyme Biotech Co., Ltd.). Image Lab (version 3.0; Bio-Rad Laboratories, Inc.) was used for densitometry.
Ecl solution
Manufactured by Vazyme
Sourced in China
ECL solution is a chemiluminescent detection reagent used for Western blotting. It is designed to detect and visualize target proteins on membranes. The solution contains the necessary components to generate a luminescent signal when exposed to the enzyme-labeled secondary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti ube2t, by Abcam (1 mentions)
Bca kit, by Bio-Rad (1 mentions)
X ray film, by Carestream (1 mentions)
Grp78, by Proteintech (1 mentions)
Image pro plus 7, by Media Cybernetics (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Tanon 5200 imaging system, by Shanghai Tanon Science & Technology (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Flotillin 1, by BD (1 mentions)
Flotillin 2, by BD (1 mentions)
Aggrecan, by Abcam (1 mentions)
Traf6, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Lamin b1, by Cell Signaling Technology (1 mentions)
8 protocols using ecl solution
1
Protein Expression Analysis Protocol
2
Protein Extraction and Western Blot
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Cell lysis buffer (CST, USA) was utilized for total protein extraction of cells. Total protein was quantified using a BCA Kit (Bio–Rad, USA) and was then separated using a 4–20% polyacrylamide gel (Willget, China). After the proteins were transferred to membranes for 1.5 h, the membranes were blocked for 1 h and then incubated with primary antibodies and secondary antibodies successively. The bands were luminesced using ECL solution (Vazyme, China) and exposed to X-ray film (Carestream, Canada).
3
Western Blot Analysis of Endoplasmic Reticulum Stress Markers
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Proteins were extracted from HEI-OC1 cells after lysis using RIPA buffer (cat. no. BL504A; Biosharp; Labgic Technology Co., Ltd.) on ice, and the protein concentration was determined using the bicinchoninic acid method and normalized. Subsequently, SDS-polyacrylamide gel electrophoresis on 6 or 8% gels was performed to separate proteins (20 µg/lane). Protein samples were then transferred to PVDF membranes (MilliporeSigma) and the membranes were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Diluted primary antibodies against CHOP (cat. no. 15204-1-AP; 1:1,000; Proteintech Group, Inc.), GRP78 (cat. no. 11587-1-AP; 1:5,000; Proteintech Group, Inc.), XBP1 (cat. no. 24168-1-AP; 1:1,000; Proteintech Group, Inc.), phosphorylated (p)-PERK (cat. no. 3179; 1:1,000; Cell Signaling Technology, Inc.), PERK (3192; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat. no. 81115-1-RR; 1:10,000; Proteintech Group, Inc.) were used to incubate the membranes at 4˚C overnight, followed by incubation with a HRP-conjugated secondary antibody (cat. no. SA00001-2; 1:5,000; Proteintech Group, Inc.) at 37˚C for 1 h. The ECL solution (Vazyme Biotech Co., Ltd.) was then added to evenly cover the membranes for 1 min, and densitometric analysis was performed using the Image-Pro Plus 7.0 software (Media Cybernetics, Inc.).
4
Protein Expression Analysis in A549 Cells
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After drug intervention or no drug intervention, A549 or A549/DDP cells were lysed and subjected to western blot analysis as a general method [16 (link)]. After treatment, the blots were probed with antibodies against Beclin 1, LC3, p62, cleaved caspase-3, p53, Bax, Bcl-2, FOXO3a, p-FOXO3a Ser253, p-FOXO3a Ser315, and p-FOXO3a Thr32; then, we detected the protein bands with ECL solution (Vazyme, Nanjing, China) using a Tanon 5200 imaging system (Tanon Science & Technology, Shanghai China) [17 (link)].
5
Western Blot Analysis of EGFR Signaling
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Cells were lysed in RIPA buffer (50 mmol/L Tris‐HCl, 150 mmol/L NaCl, 1% sodium deoxycholate, 1% Triton X‐100, 0.1% SDS and pH 7.4) supplemented with protease inhibitor cocktail (Selleck Chemicals, Houston, TX, USA) and phosphatase inhibitor cocktail (Sigma‐Aldrich). Proteins were subjected and separated on SDS‐PAGE gel and transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking with non‐fat milk or BSA, membranes were incubated overnight with the following specific primary antibodies: EGFR, phospho‐EGFR (Y1068), ERK1/2, phospho‐ERK1/2 (T202/T204), phospho‐Merlin (S518) (CST, Danvers, MA, USA), β‐tubulin, GAPDH (Sigma‐Aldrich), Merlin, caveolin‐1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), B3GALT4, Na+/K + ATPase (Abcam, Cambridge Cambridgeshire, UK), flotillin‐1 and flotillin‐2 (BD Biosciences, Franklin Lakes, NJ, USA). The membranes were then incubated with appropriate HRP‐conjugated secondary antibodies. Signals were visualized using ECL solution (Vazyme, Nanjing, China) and detected with gel documentation system (Tanon, Shanghai, China).
6
Western Blot Protein Analysis
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Cell samples were lysed with NP-40 buffer and the cell lysates were centrifuged at 12 000 g for 15 min at 4 °C. Then, the lysates were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in PBS for 1 h at room temperature, followed by washing three times with PBST. The membranes were incubated with corresponding primary antibodies and HRP-conjugated secondary antibodies. Finally, ECL solution (Vazyme) was applied to bands to measure the protein expression levels with the Tanon-5200 Chemiluminescence Imager (Tanon Science and Technology Company).
7
Evaluating Cartilage Protein Expression
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Total proteins were isolated from treated human T/C-28a2 cells using the lysis buffer and were quantified using the bicinchoninic acid (BCA) kit (Vazyme, Nanjing, China), followed by being loaded onto the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation for 1 h, proteins were transferred to the polyvinylidene fluoride (PVDF) membrane (Bio-Rad, California, USA) and the membrane was incubated with the primary antibody against type 2 collagen (1:500, #ab34712, Abcam, USA), Aggrecan (1:500; #216965, Abcam, USA), TRAF-6 (1:2000; #8028, Cell Signaling Technologies, USA), IκBα (1:3000; #4814, Cell Signaling Technologies, USA), p-IκBα (1:1000, #5209, Cell Signaling Technologies, USA), NF-κB p65 (1:2000; #3036, Cell Signaling Technologies, USA), lamin B1 (1:5000, #13435, Cell Signaling Technologies, USA), and β-actin (1:10,000, #3700, Cell Signaling Technologies, USA). Then, the membrane was incubated with the Horseradish Peroxidase (HRP)- linked goat anti-mouse (1:3000, #7076, Cell Signaling Technologies, USA) or goat anti-rabbit (1: 2000, #7074, Cell Signaling Technologies, USA) secondary antibodies, followed by 3 washes and exposure to ECL solution (Vazyme, Nanjing, China). Lastly, the quantification was conducted on the bands using the Image J software and normalized to the internal control β-actin or lamin B1.
8
Protein Quantification and Western Blot Analysis
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To obtain total proteins, cells were hydrolyzed in lysis buffer containing RIPA, PMSF, and phosphatase inhibitors (Beyotime Biotech, China). The concentration of total protein for each sample was quantitatively measured by BCA protein assay kit (Thermo, USA). Protein samples were isolated with SDS-PAGE (Bio-Rad, USA), and later transferred to PVDF membrane (Millipore, USA). And the blots were blocked using 5% of skimmed milk for 1 h, and then incubated in specific primary antibodies for 12–16 h at 4 °C. The blots were then incubated in HRP-conjugated secondary antibodies at ambient temperature. At last, protein signals were detected using ECL solution (Vazyme, China) [37 (link)]. The antibodies used in the study were displayed in Table S2.
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