For protein isolation from cells, RIPA buffer with proteinase inhibitor cocktail was used. The protein concentration was measured by BCA assay. Standard Western blot techniques and a Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA) were used according to previously described procedures [37 (link)]. The primary antibodies used were as follows: Gabra3 (1:1000, ab72446, Abcam, Cambridge, UK), p-p70s6k (1:1000, 9204, CST, Danvers, MA, USA), p70s6k (1:1000, 2708, CST), p-mTOR-S2448 (1:1000, 5536, CST), mTOR (1:1000, 2972, CST), p-AKT-S473 (1:2000, 4060, CST), AKT (1:2000, 2920, CST), MMP-9 (1:1000, 13,667, CST), MMP-2 (1:1000, 87,809, CST), p-c-Jun (1:1000, 9164, CST), c-Jun (1:1000, 2315, CST), p-JNK1/2 (1:1000, 4668, CST), JNK1/2 (1:1000, ab4821, Abcam), and β-actin (1:2000, A5316, Sigma, St. Louis, MO, USA).
Ab4821
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab4821 is a laboratory equipment product. It functions as a core component for various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ab112501, by Abcam (4 mentions)
Ab17942, by Abcam (4 mentions)
Ab9485, by Abcam (3 mentions)
Ab170099, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab6721, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab184699, by Abcam (2 mentions)
Ab38898, by Abcam (2 mentions)
Ab4822, by Abcam (2 mentions)
Ab134175, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
A5316, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab32385, by Abcam (1 mentions)
Protein extraction reagent kit, by Beyotime (1 mentions)
Ab183496, by Abcam (1 mentions)
Ab215206, by Abcam (1 mentions)
Ab169755, by Abcam (1 mentions)
Ab21981, by Abcam (1 mentions)
14 protocols using ab4821
1
Protein Extraction and Western Blot Analysis
2
Protein Extraction and Western Blot Analysis
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The protein extraction reagent kit (Beyotime, China) and 0.2% phenylmethanesulfonyl fluoride were used to obtain the proteins of cells and tissue samples. Western blotting was done with a modified version of a previous method25 (link). Anti-BUB1B (1:1000, ab183496), anti-p-c-Jun (1:1000, ab32385), and anti-p-c-Jun N-terminal kinase (JNK) (1:1000, ab4821) were purchased from Abcam. The NIH ImageJ software (National Institutes of Health, Bethesda, MD) was used to make the results visible. GAPDH and α-tubulin were used as an internal loading control.
3
Protein Isolation and Analysis in Lung Tissue
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Total proteins were isolated from right lung tissue (50 µg) and cells (5×106) using RIPA buffer (#89900; Thermo Scientific, Worcester, MA, USA) containing a protease inhibitor cocktail (#P2714; Sigma) and phosphatase inhibitor cocktail (#04906845001; Roche Applied Science, Indianapolis, IN, USA). Homogenates were centrifuged at 2,834 g at 4 °C for 20 min before the supernatant was collected. The total protein concentrations were determined by bicinchoninic acid assay (#23227, PierceTM Biotechnology, Rockford, IL, USA). Standard SDS-PAGE technique (27 (link)) was performed on equal amounts of proteins, with antibodies of p-STAT5 (ab32364; 1:500), RORγt (ab135669; 1:500), FOXP3 (ab215206; 1:500), JNK (ab112501; 1:500), p-JNK (ab4821; 1:500), arachidonate 5-lipoxygenase (Alox5; ab169755; 1:500), activator protein 1 (AP-1, ab21981; 1:500), and GAPDH (ab181602; 1:500) obtained from Abcam. The band intensity was measured by ImageJ2× 2.1.4.7 (Wayne Rasband, National Institutes of Health, USA).
4
Protein Expression Analysis Workflow
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Total protein was extracted from the cells using RIPA Lysis Buffer (Beyotime, Shanghai, China). Protein quantification was performed by bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Twelve percent SDS-PAGE (Beyotime, Shanghai; P0012A) was used to isolate the proteins, which were then transferred onto polyvinylidene fluoride (PVDF; Beyotime, Shanghai; FFP28) membranes. ECL (Amersham Pharmacia, Piscataway, NJ, USA) system was used to detect Chemiluminescence and Image J software was used for density analysis. Primary antibodies against TNF-α, Bcl-2, Bax, p-JNK, JNK and p53 were as follows: anti-TNF-α antibody (ab6671, 1:2,000, Abcam, USA), anti-Bcl-2 antibody (ab32124, 1:1,000, Abcam, USA), anti-Bax antibody (ab32503, 1:2,000, Abcam, USA), anti-P-JNK antibody (sc-6452, 1:1,000, Santa Cruz Biotechnology, USA), anti-JNK antibody (ab4821, 1:1000, Abcam, USA), anti-p53 antibody (ab131442, 1:1,000, Abcam, USA), anti-GAPDH antibody (ab9485, 1:2,500, Abcam, USA). HRP-conjugated goat anti-Rabbit IgG (ab6721, 1:2,000, Abcam, USA) were treated as secondary antibodies and antibodies against GAPDH served as an internal standard.
5
Western Blot Analysis of Cell Signaling
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Cells were lysed with RIPA lysis buffer containing protease inhibitor cocktail (GenDEPOT, Harris County, TX, USA) for 20 min on ice. The lysate was centrifuged at 12,000 rpm for 30 min at 4°C. The protein concentrations were measured using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skim milk at room temperature for 1 h, then incubated with primary antibodies overnight at 4°C. After washing extensively, the membrane was incubated with HRP-conjugated secondary antibody for 2 h. The signal was detected using enhanced chemiluminescence system.
The primary antibody information used in this study is as follows: anti-KIF2A (ab197988), anti-GAPDH (ab8245), anti-N-cadherin (ab18203), anti-p-Akt (ab38449), anti-Akt (ab8805), anti-p-ERK (ab79483), anti-ERK (ab184699), anti-p-JNK (ab4821), anti-JNK (ab112501), anti-p-c-Jun (ab32385), and anti-c-Jun (ab40766) were purchased from Abcam; anti-E-cadherin (60335-1-Ig) and anti-Vimentin (10366-1-AP) were purchased from Proteintech.
The primary antibody information used in this study is as follows: anti-KIF2A (ab197988), anti-GAPDH (ab8245), anti-N-cadherin (ab18203), anti-p-Akt (ab38449), anti-Akt (ab8805), anti-p-ERK (ab79483), anti-ERK (ab184699), anti-p-JNK (ab4821), anti-JNK (ab112501), anti-p-c-Jun (ab32385), and anti-c-Jun (ab40766) were purchased from Abcam; anti-E-cadherin (60335-1-Ig) and anti-Vimentin (10366-1-AP) were purchased from Proteintech.
6
Western Blotting Analysis of Soft Palate
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Western blotting analysis was carried out as reported previously by our lab (20 (link)). Briefly, after the total protein of soft palate tissues was extracted and separated, the samples were transferred onto a polyvinylidene fluoride (PVDF) membrane. Following washing, the membrane was incubated overnight at 4°C with primary antibodies against cAMP (1:1000, Abcam, ab76238), protein kinase A (PKA) (1:1000, Abcam, ab75991), cAMP-regulated guanine nucleotide exchange factor II (Epac2) (1:1000, Abcam, ab193665), rat sarcoma protein (Ras) (1:1000, Abcam, ab52939), c-Jun N-terminal kinase 1/2 (JNK1/2) (1:1000, Abcam, ab4821), Annexiv V (1:500, Abcam, ab14196), GAPDH (1:1000, Abcam, ab8245), and Tubulin (1:1000, Abcam, ab44928). After washing, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at room temperature, followed by color development using a mixed solution. The image analyzer quantitative system was used to quantitatively determine the intensity of the target and reference proteins.
7
Western Blotting Analysis of MAPK Signaling
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Protein was extracted using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). The protein samples were resolved by 10% SDS-PAGE and were transferred to the PVDF membrane. Next, the membranes were blocked with 5% skim milk at 25 °C for 1 h, then were incubated with the primary antibodies AEBP1 (1:1000, ab168355, Abcam), JNK1/2 (1:1000, ab112501, Abcam), p-JNK1/2 (1:1000, ab4821, Abcam), p38 (ab170099, Abcam), p-p38 (1:1000, ab178867, Abcam) and GAPDH (ab9485, Abcam) overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 25 °C. Blots were detected by enhanced chemiluminescence reagent and western blotting reagents. GAPDH was employed as a protein loading control.
8
Hippocampal Immunofluorescence Staining Protocol
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A series of 5 μm slices were cut from the paraffin-embedded slices including the hippocampus obtained on day 2. Double immunofluorescence staining was performed as previously described19 (link). Sections were incubated with primary antibodies overnight in a humidified chamber at 4 °C. Primary antibodies were rabbit monoclonal anti-p-JNK (Abcam, ab4821; 1:200), anti-neuronal nuclear antigen (Millipore, MAB377; 1:200), mouse monoclonal anti-GFAP (Cell Signaling Technology, #3,670; 1:50), and anti-Iba-1 (Abcam, ab1690; 1:200). For fluorescence staining, sections were incubated with secondary antibodies using Alexa flour-conjugated goat anti-mouse (Abcam, ab150113; 1:200) and goat anti-rabbit Cy3 (Jackson ImmunoResearch, 111-165-144; 1:200). Sections were examined using the BZ-X700 fluorescence microscope.
9
Crocin and Inflammatory Pathways
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Crocin (purity assay of ≥ 98%) was purchased from Shanghai Yuanye Biological Technology Co., Ltd. Methylthiazolyldiphenyl-tetrazolium bromide (MTT), LPS, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), fluorescein isothiocyanate (FITC)-albumin, and dimethyl-sulfoxide (DMSO) were obtained from Sigma-Aldrich. Rabbit monoclonal antibody to HMGB1 (ab79823), NF-κB p65 (ab32536), IκBα (ab32518), p-IκBα (ab133462), JNK (ab112501), ERK (ab184699), p38 (ab170099), p-ERK (ab201015), p-JNK (ab4821), p-p38 (ab47363), LaminB1 (ab133741), MMP-9 (ab38898), HPA (ab85543), SDC-4 (ab24511), Ly6g (ab25377), β-actin (ab8224), GAPDH (ab181602), MMP-9 inhibitor (MMP-9 inhib, ab142180), and FITC (ab25539) were acquired from Abcam Trading Company Ltd. Mouse polyclonal antibody to cathepsin L (CTL) was purchased from Santa Cruz Biotechnology. Mouse polyclonal antibody to HS was purchased from AMS Biotechnology (Switzerland, MA). Thrombomodulin/BDCA-3 was obtained from R&D Systems (USA). HRP-conjugated goat anti-rabbit IgG, goat anti-rabbit IgG/Alexa Fluor 594, and rabbit anti-goat IgG/Alexa Fluor were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. Cathepsin L inhibitor (CTL inhib, CAS 167498-29-5) was obtained from Santa Cruz Biotechnology.
10
Protein Extraction and Western Blot Analysis
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The total protein in cells or tissues was extracted using RIPA buffer (BOSTER, AR0105, China). The protein concentration was determined using the BCA method (KeyGEN BioTECH, KGP902, China), and 40 μg of protein from each sample was separated on 10% SDS-PAGE gels and then transferred to 0.45-µm PVDF membranes (Millipore, IPVH00010, USA) with 150 mA current for 1 h. Skim milk (5%) was used as the blocking solution and incubated with the membranes at 37 °C for 1 h. Then, the membranes were incubated with anti-MAP3K2 (1:500, ab33918, Abcam, USA), anti-PBX3 (1:1000, ab56239, Abcam, USA), anti-p-ERK1/2 (1:1000, ab214362, Abcam, USA), anti-p-p38 (1:1000, ab4822, Abcam, USA), anti-p-JNK (1:1000, ab4821, Abcam, USA), anti-ERK (1:1000, ab17942, Abcam, USA), anti-p38 (1:1000, ab31828, Abcam, USA), anti-JNK (1:1000, ab124956, Abcam, USA), anti-Cyclin A (1:1000, ab181591, Abcam, USA), anti-Cyclin D (1:1000, ab134175, Abcam, USA), anti-Bcl-2 (1:1000, ab692, Abcam, USA) or anti-Caspase-3 (1:1000, ab13585, Abcam, USA) antibody at 4 °C overnight. After 3 washes in TBST, the PVDF membrane was incubated with goat anti-rabbit IgG (1:3000, ab6721, Abcam, USA) for 1 h at room temperature. Finally, the protein bands were visualized using ECL Western Blotting Substrate (Invitrogen, 32109, USA).
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