The extracted proteins were separated by 10% SDS-PAGE and further transferred onto PVDF membranes (Millipore, Bedford, MA, United States). Antibodies including NAP1L1 (Abcam), CCND1 (Abcam), HDGF (Proteintech), and c-Jun (Proteintech) were used in the Western blot assays following the manufacturer’s instructions. Detection was performed using ECL Plus Western blotting detection reagents (Millipore, United States). The specific protein expression levels of the blots were normalized to GAPDH (Santa). The Cat numbers, origins, and dilution concentrations used for all antibodies are listed in Supplementary Table 2 .
C jun
Manufactured by Proteintech
Sourced in China, United States
C-JUN is a protein that functions as a transcription factor. It is involved in regulating gene expression and plays a role in cellular processes such as proliferation, differentiation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
C fos, by Proteintech (4 mentions)
Gapdh, by Proteintech (3 mentions)
Ecl plus western blotting detection reagents, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
C myc, by Proteintech (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Bcl 2, by Proteintech (2 mentions)
Loxl2, by Abcam (2 mentions)
β catenin, by Proteintech (2 mentions)
Nap1l1, by Abcam (1 mentions)
Ccnd1, by Abcam (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Phospho erk, by Proteintech (1 mentions)
Histone 3, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Perk t202 y204, by Cell Signaling Technology (1 mentions)
P stat5, by Cell Signaling Technology (1 mentions)
P c abl, by Cell Signaling Technology (1 mentions)
Actin hrp, by Proteintech (1 mentions)
P c jun, by Cell Signaling Technology (1 mentions)
12 protocols using c jun
1
Western Blot Protein Quantification
2
Protein Expression Analysis of Breast Cancer Cells
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Proteins from MCF-7, SKBR-3 cells and animal breast tissues were prepared using RIPA buffer containing protease inhibitor cocktail (Sigma, St. Louis, MO) and resolved on 10% SDS- polyacrylamide gels following standard protocols (Amersham BioSciences, Piscataway, NJ). Membrane were blotted with STARD10, ERBB2, ERK, phospho-ERK, c-MYC, p65, c-JUN, c-FOS (Proteintech, Rosemont, IL), control β-actin and Histone 3 (Sigma, St. Louis, MO) antibodies. Membranes were developed by chemiluminescence ECL detection system (Amersham BioSciences, Pittsburgh, PA) and blots were quantified using the Quantity OneTM densitometry program (Bio-Rad laboratories, Hercules, CA).
3
Western Blot Analysis of Signaling Cascades
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Western blot analysis was performed as previously described [24 (link)]. In brief, cell samples were counted and lysed in 1× sodium dodecyl sulfate (SDS) sample loading buffer. Equal amount of protein samples was loaded on polyacrylamide gel, followed by transfer to nitrocellulose membrane. The membrane was then blotted with specific primary antibodies against p-c-Abl (Y412, #2865S), BCR (#3902S), p-stat5 (Y694, #9351S), stat5 (#9363S), AKT (#4685S), p-AKT (Ser473, #4060S), ERK (#49655S), p-ERK (T202/Y204, #4370S), p-JNK (T183/Y185, #4668S), p-c-JUN (Ser63, #2361S) (all purchased from Cell Signaling Technology), c-Myc (#ab32072, Abcam), BRD4 (#ab128874, Abcam), JNK (#66210-1-Ig), c-JUN(#66313-1-Ig), and actin-HRP (#HRP-60008) (purchased from Proteintech). After overnight incubation at 4 °C, HRP-conjugated secondary antibodies were applied and luminescence signals on membrane were detected with electrochemical luminescence (Shanghai Share-bio Biotechnology). The western blot images were taken with a LAS4000 imaging system (Fujifilm).
4
Comprehensive Western Blot Analysis
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Whole six-well cells were lysed in 250ul 0.1% SDS and 10ul 1mM PMSF. Protein extracts were separated by SDS-PAGE and analyzed using the following primary antibodies: Id1 (Santa Cruz, sc-374287), Id3 (Santa Cruz, sc-56712), HRAS (Santa Cruz, sc-29), Vimentin (Santa Cruz, sc-6260), p-MEK (CST, 9154), p-ERK (CST, 4370), MEK (CST, 4694), ERK (CST, 4695), Snail (CST, 3879), Slug (CST, 9585), Slug (CST, 9585), MMP2 (CST, 4022), MMP9 (CST, 3852) , C-FOS (Proteintech, 66590-1-Ig), C-JUN (Proteintech, 24909-1-AP), LEF1(Abcam, ab137872), DUSP16 (Abcam, ab181088), E-cadherin(Abcam, ab40772), N-cadherin(Abcam, ab18203) and GADPH antibody (Abcam, ab8245). The next day, the membranes were incubated with secondary antibodies (CST,7076,7074) at room temperature for 2 hours. All results were repeated 3 times independently.
5
Signaling Pathways Modulation in Cells
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Sodium hydrogen sulfide (NaHS), H2O2, PD98059 (an inhibitor of ERK1/2), LY294002 (an inhibitor of PI3K) and AG1478 (an inhibitor of EGFR) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies for anti-cleaved caspase-3 and -9, Bcl-2, cytochrome c (Cyt-c), c-myc, c-jun, c-fos and GAPDH were from Proteintech (Wuhan, China). The anti-ERK1/2, p-ERK1/2, PI3K, p-PI3K, GSK-3β, p-GSK-3β, Akt, p-Akt, HB-EGF, EGFR and p-EGFR antibodies were obtained from Cell Signaling Technology (Denver, CO, USA). Assay kits for lactate dehydrogenase (LDH) and creatine kinase (CK) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Cell Counting Kit-8 (CCK-8) was obtained from Boster Bio-engineering Limited Company (Wuhan, China). The HB-EGF ELISA kit was purchased from Elabscience Biotechnology (Wuhan, China). All other chemicals were from Sigma or Santa Cruz.
6
Western Blot Analysis of Cellular Signaling
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The lysates of treated cells were reconstituted with loading buffer and run by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Nitrocellulose membranes (Millipore, USA) containing the transferred proteins were soaked in blocking buffer for 2 h and then separately incubated at 4°C overnight with the following primary antibodies: SelS (Sigma, USA), endothelial nitric oxide synthase (eNOS; Proteintech, Wuhan, China), p-c-jun (Proteintech, Wuhan, China), c-jun (Proteintech, Wuhan, China), p-p38 MAPK (Abcam, USA), p38 MAPK (Abcam, USA), inhibitory kappa B α kinase β (IKKβ, CST, USA), p-IKKβ (CST, USA), inhibitory kappa B α (IκBα, CST, USA), p-IκBα (CST, USA), NF-κB p65 (Bioworld, USA), Lamin B (Proteintech, Wuhan, China), and GAPDH (Proteintech, Wuhan, China). After washing for 30 min, the Nitrocellulose membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were subsequently treated with enhanced chemiluminescence (ECL, Thermo Scientific, USA) to develop the protein bands, and images were captured using the ChemiDoc MP Imaging System (Bio-Rad, USA). Quantitative analysis of the band intensities was performed with the Quantity One 4.52 software program (Bio-Rad, USA). Lamin B and GAPDH were used as internal controls.
7
Extraction and Immunoblotting of Proteins
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Total proteins of the collected cells were extracted in lysis buffer containing RIPA buffer (Beyotime, China), 1 mM phenylmethanesulfonyl fluoride (Beyotime, China), and 10% PhosSTOP (Roche, Switzerland). Approximately 20 μg total protein was loaded and separated by 12% SDS-PAGE, then transferred onto a polyvinylidene difluoride membrane (Millipore, USA). Subsequently, the PVDF membrane was blocked in TBS/Tween with 5% fat-free milk and incubated with the following primary antibodies: GAPDH (1:1000, Cell Signal Technology), LOX (1:1000, Abcam), LOXL1 (1:1000, Abcam), LOXL2 (1:1000, Abcam; 1:2000, Novus Biologicals), LOXL3 (1:100, Santa Cruz), LOXL4 (1:500, Abnova), c-JUN (1:1000, Proteintech), p-c-JUN (Ser73) (1:1000, Cell Signal Technology), JNK, p-JNK (Thr183/Tyr185) (1:1000, Cell Signal Technology), MMP2 (1:1000, Abcam), MMP9 (1:1000, Cell Signal Technology). The membrane was further incubated with HRP-conjugated secondary antibody (1:4000, Proteintech). Finally, the target protein band was visualized using an electrochemiluminescence kit (Thermo).
8
Western Blot Analysis of Signaling Proteins
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Cells were collected on ice (4°C), lysed using lysis buffer (Beyotime Institute of Biotechnology) and subsequently centrifuged at 12,000 × g for 30 min at 23°C to retain the supernatant. The BCA protein analysis kit (Beyotime Institute of Biotechnology) was used to determine protein concentration. Protein lysates (45 µg) were separated by SDS-PAGE (7.5%), transferred onto PVDF (EMD Millipore) membranes and blocked with 5% skimmed milk at 4°C overnight. The membranes were incubated with primary antibodies against c-Myc (1:1,000; cat. no. 10828-1-AP; ProteinTech Group, Inc.), c-Jun (1:2,000; cat. no. 24909-1-AP; ProteinTech Group, Inc.), Wisp1 (1:1,000; cat. no. 18166-1-AP; ProteinTech Group, Inc.), β-catenin (1:5,000; cat. no. 51067-2-AP; ProteinTech Group, Inc.) and GAPDH (1:5,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight at 4°C. The membranes were washed three times with tris-buffered saline with Tween-20 (for 10 min each) and subsequently incubated with HRP-conjugate goat anti-rabbit IgG secondary antibody (1:10,000; cat. no. 31460; Thermo Fisher Scientific, Inc.). Protein bands were visualized using enhanced chemiluminescence reagent (EMD Millipore) and densitometry analysis was performed using ImageJ software (Image-Pro Plus 6.0; National Institutes of Health) and the data were normalized to expression of the internal control GAPDH.
9
Tan IIA Bioactivity Evaluation
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Tan IIA (purity > 98% and molecular weight 294.34) was purchased from National Institutes for Food and Drug Control (Beijing, China). 2.943 mg Tan IIA was dissolved in 1 mL dimethyl sulfoxide (DMSO, Sigma, USA) to make a 1 × 104 μM stock solution, which was then added to the medium at the indicated concentrations. Cell Counting Kit-8 (CCK-8) was purchased from Abcam (USA). Fetal bovine serum and 0.05% trypsin were from Gibco (USA), and L15 medium was from HyClone (USA). GPER specific antagonist G-15 was from Cayman Chemical (Michigan, USA). The following antibodies were used: GPER, EGFR, ERK1/2, cyclin A2, cyclin D1, CDK2,4,6, caspase-3, lamin B1 (Abcam, USA), c-Fos, and c-Jun (Proteintech Group, USA).
10
Immunohistochemical Analysis of LOXL2 and c-JUN
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The detailed procedure of immunohistochemistry was described in our previous work [16 (link)]. Cell slides were incubated with primary antibody against LOXL2 (1:400, Abcam), c-JUN (1:50, Proteintech) at 4 °C overnight, and then reacted with secondary antibody conjugated with HRP (1:200, DingguoBio, Beijing). Staining intensity and the percentage of the stained tissues were scored by two independent observers. Photographs of four representative sites were captured under high-power magnification (× 200) by the Leica QWin Plus v3 software with identical setting parameters. The density of positive staining was measured by Image-Pro Plus v6.2 software (Media Cybernetics Inc., USA). Integrated optical density of the positive stains in each photograph was measured, and its ratio to the total area of each photograph was calculated.
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