Total proteins were extracted from cells or tissues using RIPA Lysis Buffer. Protein concentration was measured by BCA Protein Assay Kit. To examine the expression of proteins, the same amount of total proteins was loaded on an 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were transferred into PVDF membranes. After being blocked in skim milk solution, the membrane was incubated overnight separately with antibodies anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PI3K, anti-p-Akt, anti-p-Bad, anti-Cyt-c, anti-cleaved caspase 9, anti-cleaved caspase 3, anti-PARP, anti-DRP1, and anti-Mfn2 (Cell Signal, CST, USA). After that, the membrane was incubated with the secondary HRP-conjugated goat anti-rabbit antibodies (Santa Cruz Biotechnology). Proteins were visualized using an enhanced chemiluminescence kit from Thermo Fisher Scientific (Massachusetts, USA). ImageJ software (Alpha View SA) was used to perform densitometric analysis.
Anti p bad
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p-Bad is a laboratory reagent used to detect the phosphorylated form of the Bad protein. Bad is a pro-apoptotic Bcl-2 family member that plays a role in regulating cell survival and apoptosis. Phosphorylation of Bad inhibits its pro-apoptotic function.
Automatically generated - may contain errors
Lab products found in correlation
Anti p akt, by Cell Signaling Technology (5 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti p p65, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti bad, by Cell Signaling Technology (2 mentions)
Anti cyt c, by Cell Signaling Technology (1 mentions)
Anti drp1, by Cell Signaling Technology (1 mentions)
Anti mfn2, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Santa Cruz Biotechnology (1 mentions)
Anti pi3k, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Tunel staining kit, by Promega (1 mentions)
Anti p21 antibody, by BD (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Mitochondrial isolation kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti p bad
1
Protein Expression Analysis in Cells
2
PPARα Agonist Wy-14,643 Apoptosis Study
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PPARα agonist Wy-14,643 was a gift from Janardan Reddy, Northwestern University, Chicago, IL, USA. Anti-caspase-3, anti-cleaved PARP, anti-pp65, anti-pBAD and anti-BAD antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p21 antibody was purchased from BD Biosciences (San Jose, CA, USA). The TUNEL staining kit was obtained from Promega (Madison, WI, USA).
3
Mitochondrial Isolation and Protein Analysis
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Mitochondria were isolated from H9C2 cells using a mitochondrial isolation kit (Thermo Scientific) according to the manufacturer's protocol. Western blots were performed as described previously [19 (link)–21 (link)]. The membranes were incubated with appropriate primary antibodies respectively, including anti-PTEN, anti-p-Akt, anti-Akt, anti-Bim, anti-p-Bad, anti-Bad (Cell Signaling Technology, Inc, Danvers, MA), respectively, followed by incubation with peroxidase-conjugated second antibodies (Cell Signaling Technology, Inc.) and examination with the ECL system (Amersham Pharmacia, Piscataway, NJ). The signals were quantified using a G: Box gel imaging system by Syngene (Syngene, USA, Frederick, MD).
4
Localization and Quantification of Antigens in Burn Skin
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In order to determine the location and quantity of the specific antigens in the interspace skin following burn injury, the prepared slices were washed in PBS for 10 min, followed by boiling at 95°C in 0.01 mmol citrate buffer (pH, 6.0) for 10 min for antigen retrieval. Then, the slices was incubated with hydrogen peroxide for 10 min at room temperature, and 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) was applied later as the blocking solution for 20 min at room temperature. Without being washed, the sections were incubated with anti-phosphorylated (p)-Erk (1:200; cat. no. 4370; Cell Signaling Technology, Inc.) or anti-p-Bad (1:200; cat. no. sc-12969-R; Santa Cruz Biotechnology, Inc.) antibodies overnight at 4°C. After being rinsed with PBS, the sections were incubated with FITC-(1:50; cat. no. BA1105; Wuhan Boster Biological Technology, Ltd.) or Cy3-(1:50; cat. no. BA1032; Wuhan Boster Biological Technology, Ltd.) labeled goat anti-rabbit secondary antibodies for 2 h at 37°C in the dark. The sections were rinsed and stained with DAPI (100 ng/ml; Wuhan Boster Biological Technology, Ltd.) for 8 min at room temperature, and then mounted with VECTASHIELD® mounting medium (Wuhan Boster Biological Technology, Ltd.). All slices were observed and photographed under a fluorescence microscope (magnification, ×200) (DM5500B; Leica Microsystems GmbH).
5
Quantitative Protein Analysis by Western Blot
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Cells were washed with PBS and lysed with lysis buffer (Beyotime). Total protein levels were measured using a BCA protein assay kit (Pierce). Western blotting was performed as described previously [13 (link)]. Briefly, 20 μg of total protein was mixed with 2× loading buffer and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Amersham). The PVDF membranes were blocked with 5% fat-free milk dissolved in Tris-buffered saline and Tween 20 (TBST) buffer for 1 h and then incubated with the primary antibodies (anti-CIT, Abcam, ab110897; anti-CCND1, Abcam, ab16663; anti-pBad, Cell Signaling Technology (CST), #9664; anti-Bad, Abcam, ab32445; and anti-GAPDH, Santa Cruz Biotechnology, sc-32233) overnight at 4°C. After three washes with TBST buffer, the secondary antibody (Santa Cruz Biotechnology, sc-2005) was added, and immune activity was detected using an ECL-Plus kit (Amersham Biosciences).
6
Immunohistochemical Analysis of p-Akt and p-Bad
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Paraffin-embedded tissues were cut into 5-μm-thick slices for IHC examination. The sections were incubated with anti-p-Akt (1:200) and anti-p-Bad (1:200) antibodies (both from Cell Signaling Technology, Boston, MA, USA) overnight at 4 °C. Then, they were incubated with goat anti-rabbit secondary antibody (Boster, Wuhan, China), and visualized with a 3,3-diaminobenzidine (DAB) kit (Boster, Wuhan, China). Finally, the mounted sections were observed and photographed under a microscope at 200× magnification (DM2500, Leica, Solms, Germany).
7
Western Blot Analysis of Kidney Proteins
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The right kidneys of the rat models were maintained for western blotting analysis. Briefly, the frozen renal tissue samples were cut into pieces and lysed with RIPA lysis buffer (AR0105, Boster, Wuhan, China) for one hour on ice, and the lysates were centrifuged at 14,000 g for 10 min. Mixed with loading buffer, the protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes by electrophoresis, while aliquots of the coped samples were used to determine the protein concentrations of each sample with a BCA kit (KGPBCA, KeyGEN Biotech, Nanjing, China).The transferred membranes were subsequently blocked and incubated overnight at 4 °C with the following primary antibodies: anti-Akt (1:500), anti-Bad (1:1000), anti-Bcl-xL (1:800), anti-Cytochrome C (1:1000) (all from Santa Cruz, CA, USA), anti-p-Akt (1:500), anti-p-Bad (1:1000), anti-Cleaved Caspase-9 and anti-Cleaved Caspase-3 (all from Cell Signaling Technology, Boston, USA). GAPDH (Santa Cruz, CA, USA) was blotted on the same membranes as a control. Blot bands were detected with SuperSignal® West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA) and X-ray Film (Kodak, Rochester, NY, USA) and were then analysed with Bandscan 5.0 software and compared with GAPDH.
8
Oxidative Stress and Apoptosis Assay Protocol
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RMPI 1640 medium and fetal bovine serum were obtained from Hyclone (Thermo Fisher). Dimethyl sulfoxide (DMSO), 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT), phosphate buffer saline (PBS), reactive oxygen species (ROS) detection kit, lactate dehydrogenase (LDH) kit, mitochondrial membrane potential (MMP) assay kit with JC‐1, RIPA buffer, and BCA protein quantitation kit were obtained from Solabio. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total antioxidant capacity (T‐AOC), superoxidase dismutase (SOD), catalase (CAT) as well as glutathione peroxidase (GSH‐PX), and malondialdehyde (MDA) assay kits were purchased from Nanjing Jiancheng bioengineering institute. Annexin V‐FITC apoptosis detection kit, phosphatase inhibitor cocktail, nuclear protein and cytoplasmic protein extraction kit were supplied by BestBio. Anti‐β‐actin and antiproliferating cell nuclear antigen (PCNA) antibodies were provided by ABclonal. Antinuclear factor erythroid‐2‐related factor 2 (Nrf2), anti‐B‐cell lymphoma‐2 (Bcl‐2), anti‐BCL2‐Associated X (Bax), anti‐Cytochrome C (Cyt c), anti‐Caspase‐9, and anti‐Caspase‐3 were supplied by ABCAM. Anti‐Bcl‐xL/Bcl‐2 associated death promoter (Bad) and anti‐p‐Bad were purchased from Cell Signaling Technology. Anti‐Rabbit Detection Module for Wes and 12–230 kDa Wes Separation Module were purchased from ProteinSimple.
9
Protein Expression Analysis in Frozen Liver
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Frozen liver tissues were homogenized in RIPA lysis buffer in the presence of 1% (v/w) protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL). The samples were shaken at 4℃ for 1 h followed by centrifugation at 40000×g at 4℃ for another 1 h. Protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as the standard [4] . Then, 40 μg protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA) and blotted onto nitrocellulose membranes. After incubation with the specific primary antibodies anti-p65, anti-p -p-65, anti-IκBɑ, anti-bcl-2, anti-bcl-xl, anti-bax, anti-bak, anti-cytochrome c, anti-cleaved caspase 9, anti-cleaved caspase 3, anti-AKT, anti-p-AKT, anti-p38, anti-p-P38, anti-BAD, anti-p-BAD, and anti-GAPDH (Cell Signaling Technology, Danvers, MA) in blocking solution overnight at 4℃, the blots were probed with a secondary horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz, CA, USA) and developed with enhanced chemiluminescence reagents. The relative amount of the target protein was normalized to GAPDH and analyzed using a Gel Pro Analyzer (Media Cybernetics, Silver Spring, MD, USA).
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