Total cell extracts from indicated cells were obtained using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China). Protein concentration was determined by the Bicinchoninic acid (BCA) kit (Beyotime). After being separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the protein was transferred onto nitrocellulose membrane. After being blocked by 5% bovine serum albumin (BSA) for 2 h at room temperature, the membrane was incubated with primary antibodies against WNT3A (ab81614, 1 : 1000, Abcam, Cambridge, MA, USA), β‐catenin (ab32572, 1 : 2500, Abcam), or β‐actin (T0022, 1 : 10 000, Affinity, Changzhou, China) overnight at 4 °C. After three rinses, the membrane was incubated with goat anti‐mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776, 1 : 10 000, Abcam) or goat anti‐rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773, 1 : 10 000, Abcam) for 1 h at room temperature. Finally, the membrane was detected with an Odyssey infrared scanner (Li‐Cor, Lincoln, NE, USA).
T0022
Manufactured by Affinity Biosciences
Sourced in China, United States
The T0022 is a laboratory centrifuge that is designed to separate various components within a liquid sample. It operates using a spinning motion to create a centrifugal force, which causes the denser materials to move towards the outer edge of the sample, allowing the separation of different cellular or molecular components.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Affinity Biosciences (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Odyssey infrared scanner, by LI COR (1 mentions)
Ab81614, by Abcam (1 mentions)
Ab216776, by Abcam (1 mentions)
Ab216773, by Abcam (1 mentions)
Bicinchoninic acid bca kit, by Beyotime (1 mentions)
Ab32572, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by Merck Group (1 mentions)
Ab182858, by Abcam (1 mentions)
Enhanced bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ab184787, by Abcam (1 mentions)
Goat anti mouse igg, by Affinity Biosciences (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Ab49999, by Abcam (1 mentions)
Ab183597, by Abcam (1 mentions)
Ab60176, by Abcam (1 mentions)
Ab95470, by Abcam (1 mentions)
8 protocols using t0022
1
Western Blot Analysis of WNT3A and β-Catenin
2
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted with SDS lysis buffer and 1 × protease inhibitor cocktail (Roche, Germany), and the concentrations were measured by Enhanced BCA Protein assay kit (Thermo Fisher Scientific, USA). Proteins were separated by SDS-PAGE, then transferred onto a PVDF membrane (Millipore, USA), followed by blocking with 5% milk (BD, USA) in TBST (Tris‐buffered saline with Tween‐20) solution for 1.5 h at room temperature. The membrane was incubated at 4°C overnight with corresponding primary antibodies to C1QBP (Abcam, ab101267; 1:2000), XDH (Affinity Biosciences, DF8111; 1:1000), caspase-3 (Abcam, ab184787; 1:2000), bcl2 (Abcam, ab182858; 1:2000), bax (Abcam, ab32503; 1:2000) and β-actin (Affinity Biosciences, T0022; 1:3000). After washing three times in TBST, the membranes were further incubated with HRP‐conjugated goat anti‐rabbit IgG (Affinity Biosciences, S0001; 1:5000) or goat anti‐mouse IgG (Affinity Biosciences, S0002; 1:4000) secondary antibodies for 1 h at room temperature and developed by ECL Western Blotting Detection Reagents (Millipore, USA). The expression of target protein was normalized with β-actin.
3
Western Blot Analysis of Spinal Cord Proteins
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At the termination of different treatments, cells or spinal cord tissues (2.0 mm above and below the lesion, Supplementary Fig. 2 ) were collected to extract total proteins for western blot as the previously described (Wang et al. 2022 (link); Guo et al. 2020 (link)). In this study, the following specific antibodies were used: TG2 (3557, 1:1000, Cell Signaling Technology), PSR(PAB916Hu01, 1:1000, Cloud-Clone Corp.), iNOS (ab49999, 1:2000, Abcam), Arg-1 (ab60176, 1:3000, Abcam), CD86 (91882, 1:2000, Cell Signaling Technology), CD206 (60143-1-Ig, 1:2000, Proteintech), TREM2 (ab95470, 1:1500, Abcam), pNF-κB (3033, 1:2000, Cell Signaling Technology), APOE (ab183597, 1:2000, Abcam) and β-actin (T0022, 1:5000, Affinity). After primary antibodies overnight at 4 °C, the corresponding secondary antibodies were incubated based on the manufacturer’s instructions. The immunoblots were visualized using ECL kit and scanned by ChemiDoc XRS (Bio-Rad, CA, USA). Image J software was used to analyze the intensity of the bands based on the β-actin level.
4
Oligonucleotide and Antibody Utilization
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All DNA and RNA oligonucleotides were purchased from TSINGKE, Co. (Wuhan, China). Anti-NCL antibody (ab129200, Abcam, UK), anti-HCV core 1b antibody (ab2740, Abcam, UK), monoclonal mouse β-actin antibody (T0022, Affinity Bioscience, USA), monoclonal mouse GAPDH antibody (60,004-1-Ig, Proteintech, USA), goat anti-rabbit immunoglobulin G (IgG, H+L) horseradish peroxidase (HRP, S0001, Affinity Bioscience, USA), and goat anti-mouse IgG (H+L) HRP (S0002, Affinity Bioscience, USA) were used in this study.
5
Protein Expression Analysis in Liver Tissues
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Liver tissues were sonicated in ice-cold radioimmunoprecipitation lysis buffer (Beyotime, Shanghai, China) containing a phosphatase inhibitor cocktail (Beyotime, Shanghai, China) and a protease inhibitor cocktail (Beyotime, Shanghai, China). Protein concentrations were determined by a quantitative bicinchoninic acid (BCA) assay. A total of 20–30 μg of protein was used for immunoblotting. Primary antibodies against GPX4 (1:5000, ab125066, Abcam), xCT (1:1000, ab37185; Abcam), Ptgs2 (1:1000, ab15191, Abcam), TFR (1:1000, ab84036; Abcam; 10084-2-AP; Proteintech), GSK3β, P-GSK3β, Nrf2, and iNOS (1:1000, ab178945; Abcam), Nrf2 (1:1000, 16396-1AP; Proteintech), TGFβr1 (1:1000, ab31013; Abcam), CHAC1 (1:1000, 15207-1-AP; Proteintech), DHODH (1:1000, 14877-1-AP; Proteintech), FSP1 (1:1000, 20886-1-AP; Proteintech), POR (1:1000, ab180597; Abcam), caspase 8 (1:1000, 4927S; Cell Signaling Technology), cleaved caspase 8 (1:1000, 8592S; Cell Signaling Technology), caspase 9 (1:1000, 9504S; Cell Signaling Technology), BCL2 (1:1000, sc-7382; Santa Cruz Biotechnology, Santa Cruz, CA), BAX (1:1000, sc-20067; Santa Cruz Biotechnology), RIP3 (1:1000, sc-374639; Santa Cruz Biotechnology), GSDMD (1:1000, ab209845; Abcam), β-actin (1:2000, T0022; Affinity), and glyceraldehyde-3-phosphate dehydrogenase (1:2000, 2118S; Cell Signaling Technology) were applied in the study.
6
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from mouse lung tissues or cells using RIPA buffer (Beyotime) containing protease inhibitors (Beyotime). The protein concentration was evaluated with the Pierce BCA method. Then, each protein sample was separated by 8–12% SDS-PAGE, and the proteins were transferred to nitrocellulose membranes. After blocking in 5% skim milk, the membranes were probed with the following primary antibodies overnight at 4°C: anti-MAD2L1 (1 : 3,000, # A300-301A, Bethyl), anti-β-actin (Affinity), anti-Pink1 (1 : 2000, #T0022, Affinity), anti-LC3 (ab192890, # ab192890, Abcam), anti-MFN1 (1 : 1000, #14739, Cell Signaling Technology), anti-MFN2 (1 : 1000, #ab124733, Abcam), anti-Pink1(1 : 1000, #AF7581, Affinity), anti-OPA1 (1 : 1000, #DF8587, Affinity), anti-p53 (1 : 1000, #2527, Cell Signaling Technology), anti-phospho-p53 (1 : 1000, # AF3037, Affinity), anti-p21 (1 : 1000, #2947, Cell Signaling Technology), anti-phospho-p21 (1 : 1000, #AF3290, Affinity), anti-collagen1 (1 : 1000, # ab34710, Abcam), anti-E-cadherin (1 : 1000, # ab2786, Abcam), anti-N-cadherin (1 : 1000, #13116, Cell Signaling Technology), and anti-Vimentin (1 : 1000, # BF8006, Affinity). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots were visualized by using an enhanced chemiluminescence system (Bio-Rad, USA).
7
Cell Culture and Knockdown of FOXQ1
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The THP-1 cell line and the human GC cell lines, MKN45 and MKN74, were used in the present study. All cell lines were obtained from the Cell Bank of Shanghai (Shanghai, China). The cells were grown in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) that was supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml), and were incubated in a humidified atmosphere containing 5% CO2 at 37°C and the medium was replaced three times/week.
Rabbit anti-FOXQ1 (1:100; ab51340; Abcam, Cambridge, MA, USA), rabbit anti-E-cadherin (1:100; AF0131; Affinity, Sterling, VA, USA), rabbit anti-vimentin (1:100; 5741; Cell Signaling Technology, Inc., Beverly, MA, USA) and mouse anti-β-actin (1:100; T0022; Affinity) were used as primary antibodies. The FOXQ1 shRNA lentiviral particle containing FOXQ1 shRNA sequences was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MKN45 and MKN74 cells were infected with shFOXQ1 lentiviral particles and a negative control for 48 h and followed by 2 mg/ml of puromycin selection.
Rabbit anti-FOXQ1 (1:100; ab51340; Abcam, Cambridge, MA, USA), rabbit anti-E-cadherin (1:100; AF0131; Affinity, Sterling, VA, USA), rabbit anti-vimentin (1:100; 5741; Cell Signaling Technology, Inc., Beverly, MA, USA) and mouse anti-β-actin (1:100; T0022; Affinity) were used as primary antibodies. The FOXQ1 shRNA lentiviral particle containing FOXQ1 shRNA sequences was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MKN45 and MKN74 cells were infected with shFOXQ1 lentiviral particles and a negative control for 48 h and followed by 2 mg/ml of puromycin selection.
8
Western Blot Analysis of Cellular Proteins
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Tissue was lysed in RIPA buffer lysis system (Epizyme, Shanghai) supplemented with a proteinase inhibitor cocktail (Epizyme, Shanghai) on ice. The supernatant was collected to assess for total protein using BCA Protein Assay. Equal amounts of protein (20 μg) were loaded onto sodium dodecyl sulfatepolyacrylamide electrophoresis gels. Separated proteins in gels were transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were blocked in 5% nonfat milk or bovine serum albumin (BSA) for 1 h and subsequently probed overnight at the temperature of 4 °C with diluted primary antibodies against β-Actin (1:5000, T0022, Affinity biosciences, ChangZhou, China), UBA1(1:2000, ab180125, Abcam Inc, Cambridge, UK), EIF3E (1:1000, ab134958, Abcam Inc, Cambridge, UK), RPL27 (1:500, 14980-1-AP, Proteintech, Chicago, USA), RPL17 (1:5000, 67223-1-Ig, Proteintech, Chicago, USA), RPS28(1:500, 14796-1-AP, Proteintech, Chicago, USA). Membranes were washed in wash buffer (1 × TBS 0.05% Tween-20). Following incubation with HRP-conjugated secondary antibodies for 1 h at room temperature, enhanced chemiluminescence reagent (Epizyme, Shanghai) was applied to develop the signal, which was detected by a ChemiDoc CRS imaging system (Bio-Rad, USA) [30 ].
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