Tissue and cell samples were lysed in RIPA buffer containing a protease inhibitor cocktail. Protein concentration in the lysed sample was detected using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, USA). After that, samples containing 5× load buffer were heated for 5 min at 95°C. Equal amounts of protein were separated by 4-20% SDS-PAGE, and transferred to PVDF membranes (BIO-RAD, USA). Then, the membranes were blocked with 5% non-fat milk in TBS with 0.1% Tween-20 for 1 h and incubated with rabbit anti-GAPDH (Abcam, 1:10000), rabbit anti-lysozyme (Abcam,1:1000) overnight, respectively. After the washing, goat anti-rabbit secondary antibodies (Bioss, 1:1500) were used to incubate the membranes. Finally, the optical protein bands were developed using efficient chemiluminescence (ECL) kit, and light emission was captured using the Versa DOC 4000 imaging system.
Goat anti rabbit secondary antibody
Manufactured by Bioss Antibodies
Sourced in China
Goat anti-rabbit secondary antibody is a lab equipment product used to detect the presence of rabbit primary antibodies in various immunoassays. It serves as a detection tool by binding to the rabbit primary antibody, allowing for the visualization and quantification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (3 mentions)
Rabbit anti gapdh, by Abcam (2 mentions)
Bca assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Rabbit anti lysozyme, by Abcam (1 mentions)
Ecl chemiluminescent detection system, by Thermo Fisher Scientific (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
P pi3k, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Rabbit anti tlr4, by Abcam (1 mentions)
Protein a agarose sc 2001, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Non fat dry milk powder, by Merck Group (1 mentions)
10 protocols using goat anti rabbit secondary antibody
1
Protein Quantification and Western Blotting
2
Examining Protein Signaling Pathways
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Cell protein lysates were extracted from cells using 1× sodium dodecyl sulfate buffer and quantified by Bicin Choninic Acid (BCA) protein assay kit (Thermo, Waltham, MA, USA). On a 10% SDS-PAGE (Sodium doecylsulfate-polyacrylamide gel electrophoresis), 30 μg of protein samples per lane were separated and then electransferred to a PVDF (polyvinylidene fluoride) membrane. The samples were blocked in 5% nonfat milk powder and then incubated with antibodies against PTEN (Abcam, Burlingame, CA, USA), AKT, p-AKT, PI3K, p-PI3K,GSK3β, p-GSK3β, Bax, Bcl-2, Caspase-3 cleaved (Cell Signaling Technology, Beverly, MA, USA), and β-actin (Proteintech, Rosemont, IL, USA) at 4 °C overnight. Blots were incubated with goat anti-rabbit secondary antibodies (Bioss, Beijing, China) for 2 h at room temperature. β-actin was used as an internal control. The bands were visualized by an ECL chemiluminescent detection system (Thermo Scientific, New York, NY, USA).
3
Protein Expression Analysis in Tissue Samples
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Tissue samples and organoids were lysed in RIPA buffer containing a protease and phosphatase inhibitor cocktail. Protein concentrations of the lysed samples were detected using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, Waltham, MA, USA), after which samples containing 5× load buffer were heated at 95 °C for 5 min. Equal amounts of protein in various samples were separated by 4–20% SDS-PAGE and transferred to PVDF membranes (BIO-RAD, Hercules, CA, USA). After that, the membranes were blocked with 5% non-fat milk in TBS with 0.1% Tween-20 for 1 h and then incubated with rabbit anti-GAPDH (abcam, Cambridge, UK, RRID: AB_2107448), rabbit anti-CCN1(1:1000, Affinity, Cincinnati, OH, USA, RRID: AB_2838216), rabbit anti-TLR2 (1:1000, Affinity, Cincinnati, OH, USA, RRID: AB_2838958), rabbit anti-TLR4 (1:1000, abcam, Cambridge, UK, RRID: AB_300696), rabbit anti-p38 (1:1000, CST, Beverly, MA, USA, RRID: AB_10999090) and rabbit p-p38 (1:1000, CST, Beverly, MA, USA, RRID: AB_331641) overnight. After washing, goat anti-rabbit secondary antibodies (Bioss, 1:1000) were used to incubate the membranes for 2 h. Finally, the optical protein bands were developed using the efficient chemiluminescence (ECL) kit, and light emission was captured using the Versa DOC 4000 imaging system.
4
Immunohistochemical Detection of BMP-7
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Immunostaining was performed using a standard protocol. The sections were deparaffinized, subjected to 0.25% trypsin at 37 °C for 30 min to retrieve the antigen, treated with 3% H2O2 to inhibit endogenous peroxidase activity at room temperature for 20 min, blocked with 10% goat serum at room temperature for 30 min, and then incubated with rabbit polyclonal anti-BMP-7 antibody (1:100; Wanlei, Shenyang, China) overnight. On the second day, the sections were incubated with goat anti-rabbit secondary antibody (1:100, Bioss, Beijing, China). The color was developed using a diaminobenzidine substrate chromogen system (Bioss, Beijing, China). The cells expressing BMP-7 were stained brown and negative cells purple. Corresponding nonimmune IgGs were used as negative controls.
5
Gimap5 Interactome Identification in Cell Lines
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The Beas-2b and PC9 cells with Gimap5 overexpression were first lysed with the lysis buffer (50mM Tris HCl, pH7.4, 150mM NaCl, 1mM EDTA, and 1% Triton X-100). The extracted protein samples were then co-immunoprecipitated with rabbit anti-Gimap5 (10 μg) and IgG (BIOSS, Beijing, China), respectively. Rabbit-anti M6PR (10 μg) was subsequently added to perform secondary immunoprecipitation. Afterward, the target proteins and their interacted proteins were co-immunoprecipitated by Protein A-Agarose (SC-2001, SANTA CRUZ, California, USA), and the unbound proteins were eliminated three times using PBS elution. The collected samples were then isolated using SDS-PAGE 12% bis-Tris protein gel. Next, they were transferred to the PVDF membrane and tested with the goat-anti-rabbit secondary antibody (BIOSS, Beijing, China). Finally, the remaining samples were used for tandem mass spectrometry analysis, which was performed by Jingjie Biotechnology (PTM.BIO Lab, Zhejiang, China).
6
Quantitative Western Blot Analysis
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Total protein was extracted from the cells using RIPA lysis buffer (Beyotime Ltd., Beijing, China), quantified using a BCA protein assay kit (Bio-Rad Laboratories, Hercules, USA) and separated via a 12% SDS-PAGE (NuPAGE; Invitrogen; Thermo Fisher Scientific, Inc.). The separated proteins were subsequently transferred onto a PVDF membrane (Whatman plc; GE Healthcare Life Sciences) and blocked with 5% non-fat dry milk powder (Merck KGaA) at room temperature for 2 h. The PVDF membrane was then incubated with RCBTB1 (1:1000, Immunoway, YT0974), SLC1A5 (1:1000, Immunoway, YT7482), WNT7B (1:1000, Immunoway, YN0288) and Tubulin (1:5000, Abcam, ab7291) primary antibodies at 4°C overnight. Following the primary antibody incubation, the membranes were washed with TBS-Tween 20 three times and incubated with a goat anti-rabbit secondary antibody (1:3000, Bioss, Beijing China) for 2 h at room temperature. Protein bands were visualized using a chemiluminescence reagent on a gel imaging system. The gray value of the target protein was normalized to the gray value of the internal reference protein. Tubulin and the data are presented as the relative content of the target protein in a sample.
7
Iso-suillin Induces Apoptosis Pathway
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Cells were treated with iso-suillin at concentrations of 6.82, 13.63, and 20.45 μmol/L for 48 h. Then, cells were collected for protein extraction. Total protein was lysed using radio immunoprecipitation assay lysis buffer. The protein used for cytochrome c detection was extracted from cytosol using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China).
Antibodies (Cell Signaling Technology, USA) for β-actin, cytochrome c, cleaved caspase-9, Fas-associated protein with death domain (FADD), cleaved caspase-8, caspase-3, Bcl-2, and Bax were incubated overnight at 4°C. The membrane was then incubated with goat anti-rabbit secondary antibody (Bioss, China) at room temperature for 60 min. Enhanced chemiluminescence kit (CWbio, China) was used before analyzing with a chemiluminescence imaging system (Image Reader Las-4000, Fuji, Japan).
Antibodies (Cell Signaling Technology, USA) for β-actin, cytochrome c, cleaved caspase-9, Fas-associated protein with death domain (FADD), cleaved caspase-8, caspase-3, Bcl-2, and Bax were incubated overnight at 4°C. The membrane was then incubated with goat anti-rabbit secondary antibody (Bioss, China) at room temperature for 60 min. Enhanced chemiluminescence kit (CWbio, China) was used before analyzing with a chemiluminescence imaging system (Image Reader Las-4000, Fuji, Japan).
8
Protein Expression Analysis via Western Blot
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RIPA lysis buffer (Beyotime, Shanghai, China) was used for protein extraction, and the BCA Protein Assay Kit was used to measure the amount of protein (Solarbio, Beijing, China). Total proteins were then subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electrotransferred to PVDF membranes (Bio-Rad, Hercules, CA, United States). Nonspecific binding sites were blocked using 5% nonfat milk for 2 h and the membranes were incubated at 4 °C overnight with rabbit anti-Wnt2 antibody (1:1000; Affinity, Melbourne, United States), rabbit anti-c-myc antibody (1:1000; Bioss, Beijing, China), rabbit anti-CD44 antibody (1:1000; Affinity, Melbourne, United States), rabbit anti-cyclin D1 antibody (1:1000; Abcam, Cambridge, United Kingdom), and rabbit anti-β-tubulin antibody (1:4000; Proteintech, Chicago, United States). The PVDF membranes were then treated for 1 hour with a goat anti-rabbit secondary antibody that was HRP conjugated (1:5000; Bioss, Beijing, China). Enhanced chemiluminescence reagents (Beyotime, Shanghai, China) were used to visualize the bands. Image J was utilized to quantify the chemiluminescent signals of protein bands using β-tubulin as an internal control.
9
Western Blot Analysis of GLUT4, TLR4, and NF-κB
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Tissue lysates were prepared in RIPA buffer (Beyotime, Shanghai, China) according to a previously described method (Luo et al., 2019 (link)). The protein concentration of each lysate was then measured using a BCA kit (Beyotime), and lysates containing equal amounts of protein were separated by SDS-PAGE (8–10%) electrophoresis and then transferred to PVDF membranes (Merck, Darmstadt, Germany). The membranes were blocked using 5% dried non-fat milk powder diluted in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and then incubated overnight at 4°C with the appropriate primary antibody. The next day, the membranes were incubated with the appropriate secondary antibody for 1 h. Specific bands were visualized using the GelView 6000Plus smart imaging system (Boluteng, Guangzhou, China). The antibodies used for western blotting were as follows: anti-β-actin (1:2,000, Bioss, Beijing, China), goat anti-rabbit secondary antibody (1:5,000, Bioss), goat anti-mouse secondary antibody (1:5,000, Beyotime), anti-GLUT4, anti-Toll-like receptor 4 (TLR4), and anti-nuclear factor kappa-B (NF-κB) (1:2,000, Cell Signaling Technology, Boston, MA).
10
Antibody-based Evaluation of PI3K/Akt Signaling
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Antibodies included rabbit anti-Akt1 (1:1000, CST: #75,692, USA), rabbit anti-p-Akt1 (1:1000, CST: #9018, USA), rabbit anti-FoxO3a (1:1000, CST: #2497, USA), rabbit anti-p-FoxO3a (1:1000, CST: #9466, USA), rabbit anti-Bim (1:1000, CST: #2933, USA), rabbit anti-GAPDH (1:1000, CST: #2118, USA) and goat anti-rabbit secondary antibody (1:3000, Bioss biotechnology Co. LTD., China). Others included Total RNA isolation kit (Tiangen Biotech Co., LTD., China), TRIzol reagent Kit, SYBR Premix Ex Taq II enzyme kit, and PrimeScriptRT Master Mix (Takara Bio, Inc., China), Evans blue, 2,3,5-triphenyltetrazolium chloride (TTC, Solarbio Technology, China), LY294002 (PI3K inhibitor, Sigma), Lactate dehydrogenase (LDH) and Creatine kinase (CK) Kits (MedicalSystem Biotechnology CO. Ltd, China), goat anti-rabbit IgG/FITC (Beijing biosynthesis biotechnology CO.Ltd, China), Annexin V-FITC Apoptosis Detection Kit(Beyotime biotechnology, China) and BCA protein Assay kit (Merck Millipore Technology, Germany). Oligo synthesis was performed by Sangon Biotech co. LTD (Shanghai, China).
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