The lung tissues harvested from mice that underwent different treatments were homogenized in PBS and radioimmunoprecipitation assay (RIPA) buffer. The homogenates were centrifuged at 12,000×g for 15 min to obtain the respective supernatants. Protein samples (50 μg) were resolved using denaturing 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods, and were after transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were subsequently incubated with a blocking solution of 5% non-fat milk for 1.5 h at room temperature. The blotted proteins were separately probed with the indicated primary antibodies at 4 °C overnight or at room temperature for 2 h. The membranes were subsequently incubated with horseradish peroxidase-conjugated (HRP) secondary antibody at room temperature for 1 h, and the immuno-labeled proteins were visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories). The following antibodies were used: rabbit anti-mouse iNOS polyclonal antibody (ab3523, Abcam), rabbit anti-mouse COX-2 polyclonal antibody (ab15191, Abcam), rabbit anti-mouse beta-actin antibody (ab189073, Abcam), rabbit anti-mouse NF-κB p65 polyclonal antibody (ab16502, Abcam), rabbit anti-mouse IκB alpha polyclonal antibody (ab32518, Abcam). HRP-conjugated goat anti-rabbit IgG (ab6721, Abcam) was used as secondary antibody.
Ab189073
Manufactured by Abcam
Sourced in United States
Ab189073 is a polyclonal antibody that targets the protein GFP (green fluorescent protein). This antibody is designed for use in immunohistochemistry, western blotting, and other applications requiring the detection of GFP-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting substrate, by Promega (2 mentions)
Lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab32518, by Abcam (1 mentions)
Ab3523, by Abcam (1 mentions)
Ab15191, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ab81046, by Abcam (1 mentions)
Bio image system, by Bio-Rad (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Proteinase and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Ab91526, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Ab77302, by Abcam (1 mentions)
L7543, by Merck Group (1 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (1 mentions)
6 protocols using ab189073
1
Protein Expression Analysis of Lung Tissues
2
Quantification of LIMK1 and Phospho-LIMK1
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Isolated left hippocampus tissue was lysed in 100 µL radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Haimen, Jiangsu, China) plus protease inhibitors. Total protein (50 µg) was loaded into 10% SDS-PAGE gels, electrophoresed, and then transblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA) in a Tris-glycine transfer buffer. After being blocked in 5% milk in PBST for 1 h at room temperature with shaking, membranes were incubated with antibodies overnight at 4°C against the following: anti-LIMK1 antibody (dilution, 1 : 1000; ab81046, Abcam) and anti-LIM kinase 1 antibody (phospho-Thr508) (dilution, 1 : 1000; ab131341, Abcam) and β-actin (dilution, 1 : 1000; ab189073; Abcam). The following day, membranes were incubated in 5% milk (in TBST) with an anti-goat or anti-mouse IgG antibody (dilution, 1 : 5000; PerkinElmer Life Sciences, Waltham, MA) for 1 h at room temperature with shaking. Membranes were washed a minimum of four times (10 min per wash) in PBST between each antibody treatment. Detected bands were visualized using enhanced chemiluminescence and images were captured using a Bio-Image system (Bio-Rad Laboratories, Inc., Hercules, USA). Western blotting was repeated three times.
3
Western Blot Analysis of Autophagy Markers
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Cell samples were lysed using a lysis buffer (Beyotime, Shanghai, China) supplemented with proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich). The protein concentration was determined using a BCA protein assay kit (Beyotime). The proteins were separated on 10% SDS PAGE gel and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with primary antibodies overnight at 4°. The primary antibodies used were rabbit anti-LC3B (L7543, 1:1000, Sigma-Aldrich), anti-SQSTM1/p62 (ab91526, 1:1000, Abcam, Cambridge, MA, USA), anti-cleaved PARP (#9546, 1:2000, Cell Signaling, Danvers, MA, USA), anti-HMGB1 (ab77302, 1:1000; Abcam), and anti-β-actin (ab189073, 1:1000; Abcam). After washing 3 times, the membranes were incubated in HRP-conjugated secondary antibodies for another 1 h at room temperature. Then, the protein signals were detected using the ECL Western blotting substrate (Promega, Madison, WI, USA).
4
Western Blot Analysis of Autophagy Markers
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Cells were lysed using a lysis buffer (Beyotime, Shanghai, China) and then protein concentration was quantified using a BCA protein assay kit (Beyotime). Then the protein samples were subjected to a conventional western blot analysis. After separation on 10% SDS PAGE gel, the proteins were subsequently transferred onto PVDF membrane and blocked with 5% nonfat milk for 1 h. Membranes were probed overnight at 4°C with primary antibodies: anti‐LC3B (1:3000, ab51520, Abcam, Cambridge, MA, USA), anti‐p62/SQSTM1 (1:1000, #8025, Cell Signaling), anti‐DICER (1:200, H‐212; Santa Cruz Biotechnology), anti‐active caspase‐3 (1:1000, ab2302, Abcam), anti‐tubulin (1:1000, ab56676, Abcam), and anti‐β‐actin (ab189073, 1:1000, Abcam). Band signals were visualized using HRP‐linked secondary antibodies (Abcam) and the ECL Western blotting substrate (Promega, Madison, WI, USA).
5
STZ-Induced Diabetic Rat Model Protocol
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Three-month-old male Wistar rats with a body weight of 200–220 g were purchased from Shanghai Slaccas Experimental Animal Co., Ltd. (Shanghai, China; Certificate no. SCXK 2015-0012). Moxibustion was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All of the rats were provided free access to water and food and maintained in a 12 h:12 h light/dark cycle at 22 ± 2 °C and 65–69% relative humidity for 8 weeks. This study was approved by the ethics committee of Hubei University of Chinese Medicine (Wuhan, China). The animal research protocol was conducted in accordance with the European Community guidelines for the use of experimental animals. STZ was purchased from Hangzhou Baitong Biological Technology Co., Ltd. (Hangzhou, China). IL-1β, IL-6, and IL-8 ELISA commercially available kits (R&D Systems, Minneapolis, MN, USA) were used. Rabbit antibody against β-actin (ab189073), rabbit anti-NF-κB polyclonal antibody (ab7971), and rabbit anti-Nrf2 polyclonal antibody (ab31163) were purchased from Abcam (Cambridge, MA, USA). Total RNA was extracted from freshly frozen neural tissues by using an Ultrapure RNA kit (CWbio Co., Ltd., China) and then reverse-transcribed with a HiFi-MMLV cDNA kit (CWbio Co., Ltd., China). Real-time PCR was performed in a Bioer line gene PCR instrument (BIOER, China) by using Invitrogen primers.
6
Quantification of Gene Expression by RT-qPCR
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Total RNA (tRNA) was isolated from cell lines using the RNeasy Mini Kit (Cat: 74104) (QIAGEN, Hilden, Germany). Reverse transcription PCR (RT-PCR) was performed using Superscript IV Reverse Transcriptase (Cat: 18090010) (ThermoFisher, Waltham, MA, USA). Triplicate samples and their corresponding controls were evaluated by qPCR. The 2-ΔΔCt algorithm determined gene fold changes. Human β-actin (1:500, ab189073, abcam) was used as a reference gene to normalize 2-ΔΔCt-based assessments, and GES-1 was used to normalize expressions between cell lines. The relative expression of genes was normalized to human β-actin. All primer sequences used are listed in Table 2 .
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