Western blot analysis was performed as previously described [14 (link)]. In brief, whole cell or tissue extracts were prepared using RIPA buffer. After electrophoresis, proteins were electroeluted at 120 Volts onto a polyvinylidenedifluoride (PVDF) membrane (Invitrogen). Indicated primary antibodies were used. Protein bands were visualized by an enhanced chemiluminescence assay kit (Super Signal Pierce Bio-technology). The following antibodies against ATGL/PNPLA2(Cayman, 10006409), PPARα(Abcam, ab8934), MAGL(Abcam, ab24701), HSL(Abcam,ab45422), p53(Santa Cruz, SC126) and p21(Abcam, ab109520), Bax (Abcam, ab32503), β-Actin (Cell Signaling Technology) were used. Western blot analyses were repeated at least three times.
Ab32503
Manufactured by Cell Signaling Technology
Sourced in United States
Ab32503 is a research-use only antibody product offered by Cell Signaling Technology. The core function of this product is to detect the target protein through immunodetection techniques. Further details on the intended use or performance characteristics are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab32124, by Cell Signaling Technology (3 mentions)
Ab182858, by Abcam (2 mentions)
Ab13847, by Cell Signaling Technology (2 mentions)
Ab189491, by Cell Signaling Technology (2 mentions)
G1214, by Wuhan Servicebio Technology (2 mentions)
Gapdh, by Wuhan Servicebio Technology (2 mentions)
G1213, by Wuhan Servicebio Technology (2 mentions)
Ab8934, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab109520, by Abcam (1 mentions)
Sc126, by Abcam (1 mentions)
Ab24701, by Abcam (1 mentions)
Ab274376, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Solarbio (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Ab189524, by Cell Signaling Technology (1 mentions)
Ab92494, by Cell Signaling Technology (1 mentions)
Ab181557, by Cell Signaling Technology (1 mentions)
12 protocols using ab32503
1
Western Blot Analysis of Lipid Metabolism and Apoptosis
2
Western Blot Analysis of Apoptosis Markers
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Western blotting was performed as per the previously published procedure [26 (link)]. Total protein from the cells was lysed using RIPA buffer (Santa Cruz Biotechnology, USA). Approximately 20 μg of protein extract was loaded onto 12% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were then exposed to 5% skim milk and probed with primary antibodies against LAMC2 (1:1000; ab274376; Abcam), B-cell lymphoma (Bcl)-2 (1:1000; ab32124), Bcl-2-associated X (Bax) (1:1000; ab32503), and GAPDH (1:1000; #5174; Cell Signaling Technology Inc., USA) at 4°C overnight, followed by examination with the corresponding horseradish-peroxidase-conjugated secondary antibodies (1:1000; SA205; Solarbio, China) at 37°C for 1 h. ECL kit (Najm Biotech ECL, Iran) was used to treat the membranes.
3
Western Blot Analysis of Stem Cell and Apoptosis Markers
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Total protein was isolated using a radioimmunoprecipitation assay lysis buffer. The extracted proteins were subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% milk and incubated with the primary antibodies at 4 °C overnight, followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies. The signals were detected using an enhanced chemiluminescence detection kit (Cell Signaling Technology, Danvers, MA, USA). The primary antibodies used were antibodies against Bach1 (1:1500, ab180853), CD44 (1:1500, ab189524), Sox2 (1:1500, ab92494), Nanog (1:1500, ab109250), Oct4 (1:1500, ab181557), p53 (1:1500, ab26), BCL2 (1:1500, ab32124), BAX (1:1500, ab32503), p-p38 MAPK and total p38 MAPK (Cell Signaling Technology, Boston, MA, USA), p-AKT1 (1:1500, phospho S473, ab81283), AKT1 (1:1500, ab28422), c-Fos (1:1500, ab190289), c-Jun (1:1500, ab32137 and GAPDH (1:1500, ab8245),all of which were obtained from Abcam.
4
Protein Analysis Using Western Blotting
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Cells and tissue were first lysed with RIPA buffer (Beyotime Biotechnology, Shanghai, China) at room temperature for 1 h. The bicinchoninic acid method was used to measure the level of proteins. Proteins in the lysate were separated by electrophoresis and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skimmed milk powder (Sigma-Aldrich) and then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: anti-MEX3C (Cell signaling, #50,844, 1:1,000), anti-RUNX3 (Cell signaling, #9647, 1:1,500), anti-Suv39H1 (Novus, NBP1-21,367, 1:1,000), anti-E-cadherin (Abcam, ab231303, 1:1,500), anti-N-cadherin (Abcam, ab76011, 1:1,000), anti-Bcl-2 (Abcam, ab182858, 1:1,000), anti-Bax (Abcam, ab32503, 1:1,000), anti-Cleaved-caspase-3 (Cell signaling, #9661, 1:1,000), and anti-GAPDH (Abcam, ab8254, 1:2,000). The membranes were then washed in TBST and incubated with horseradish peroxidase-labeled secondary antibody. After washing in TBST again, protein bands were visualized using an ECL detection agent (GE Healthcare, Chicago, IL, USA) and ImageJ software. GAPDH were used as internal loading controls.
5
Western Blot Analysis of Apoptosis Markers
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The procedure was performed partly as described previously [22 (link)]. Briefly, the protein lysis solutions were separated through gel electrophoresis and transferred onto PVDF membranes. The membranes were incubated with primary antibodies against bcl2 (Abcam, ab182858), bax (Abcam, ab32503), caspase 3 (Cell Signaling Technology, #9662), cleaved-caspase3 (Cell Signaling Technology, #9664), β-tubulin (Abbkine, #A01030), and β-actin (Abbkine, A01010) in a 4°C refrigerator overnight. Then, the membranes were coincubated with fluorescent secondary antibodies. The Odyssey imaging system was used to analyse protein expression.
6
Western Blot Analysis of Apoptosis Markers
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Total protein was extracted with the use of radio‐Immunoprecipitation Assay lysate buffer (R0010; Beijing Solarbio Science & Technology Co., Ltd. Beijing, China). Protein concentration of each sample was determined by using a BCA kit (GBCBIO Technologies, Guangzhou, Guangdong, China). The protein was separated by polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore) and sealed with 5% BSA at room temperature for 1 h. Primary rabbit antibodies (1:1000) from Abcam to cleaved‐Caspase3 (ab49822), Caspase3 (ab13847), B cell lymphoma‐2 (Bcl‐2; ab196495), Bcl‐2‐associated X (Bax; ab32503), FOS (ab190289) and p65 (ab16502) as well as primary rabbit antibody to p‐p65 (Ser536) (1:1000; 3033, Cell Signaling Technology) were added to the membrane and incubated overnight. The following day, the membrane was incubated with goat anti‐rabbit IgG (ab97051, 1:2000, Abcam) at room temperature. The immunocomplexes on the membrane were visualized using enhanced chemiluminescence reagent and imaged using Image Quant LAS 4000C gel imager (GE, General Electric Company, Schenectady, NY, USA). With rabbit anti‐β‐actin (1:3000; Abcam, ab8227) serving as an internal reference, the grey value of each band was analysed by gel image analysis software Image J.
7
Western Blot Analysis of Pino's Mechanisms in PIHF
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We implement western blot to elucidate the underlying mechanisms by which Pino ameliorated PIHF. The procedures of protein sample extraction and preparation resembled those previously reported in our study (Chen et al. 2020 (link)a). The membranes were probed with antibodies against the nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1; 1:2000, abcam, ab189491), p53 (1:1000, cell signaling technology, 2524S), bcl-2 (1:1000, cell signaling technology, 2870P), bax (1:1000, abcam, ab32503), cleaved caspase-3 (1:1000, cell signaling technology, 9664 T), collagen I (1:1000, Affinity, AF7001), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Servicebio, GB12002), with subsequent visualization by HRP-labeled secondary antibodies (Servicebio, G1213 and G1214) and chemiluminescence detection reagents. The blot images were finally analyzed and quantified by Image J software.
8
Protein Expression Profiling in Cell Signaling
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Total protein was isolated using RIPA lysis buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes. The membrane was and probed with diluted primary rabbit antibodies against Caspase-3 (ab13847), Bcl-2-associated X protein (Bax; ab32503), B-cell lymphoma 2 (Bcl-2) (ab32124), Bcl-XL (ab32370), TGF-β1 (ab92486), β-actin (ab8227), GAPDH (ab181602), E-cadherin (3195), N-cadherin (13116), matrix metalloproteinase (MMP)3 (14351), MMP9 (13667), Smad2 (5339), Smad3 (9523), phosphorylated (p)-Smad2 (18338), and p-Smad3 (9520, Cell Signaling Technologies) overnight at 4°C. The antibodies were from Abcam except for p-Smad2/3. After washing, membrane was re-probed with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab205719, 1: 2000, Abcam) for 1 h. The protein bands were visualized using enhanced chemiluminescence (EMD Millipore). β-actin and GAPDH were used as internal controls. The gray values were analyzed with Image J software.
9
Protein Expression Analysis by Western Blot
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Western blotting was performed to analyze the protein expression of Bcl-2 (Abcam, ab16904, 1:1000), Bax (Abcam, ab32503, 1:1000), p-mTOR (Cell Signaling Technology, 5536S, 1:1000), LC3B (Abcam, ab192890, 1:500), P62 (Abcam, ab51416, 1:800), DNM2 (Invitrogen, PA1-661, 1:800) and β-actin (Abcam, ab6276, 1:5000) following protein concentration measurement using the BCA protein assay reagent (Thermo Fisher Scientific, 71285). Proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 10% no-fat milk fat at room temperature for 1 h before incubation with primary antibodies at 4℃ overnight. Afterward, the membranes were rinsed and incubated with secondary antibodies for 2 h at RT. Bands were then detected by a super signal chemiluminescence detection kit (Sigma-Aldrich, S0500) and images were obtained with the ChemiDoc Imaging System (Bio-Rad, US). Band intensities were quantitated by Image Pro Plus 6.0 software and protein expression was normalized to that of β-actin.
10
Quantitative Protein Analysis in HeLa Cells
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Total protein concentration was extracted from HeLa cells using a RIPA lysate (89901, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured using a protein detection kit (A53227, Thermo Fisher Scientific, Waltham, MA, USA). A volume of 30 μg protein for each cell was transferred to a PVDF membrane (HVLP04700, Millipore, Billerica, MA, USA) using the SDS-PAGE method. After the transfer was completed, the membrane was soaked from bottom to top with TBS, then placed in an incubation box containing a 5% skimmed milk powder solution (37°C, 1 h), incubated with shaking at room temperature on a decolorization shake flask for 2 hours, and then incubated with phosphorylated (p)-Akt (1: 1000; 56 KD; ab38449; Abcam, Cambridge, UK), Akt (1: 500; 55 KD; ab8805), Bax (1: 1000; 21 KD; ab32503), Bcl-2 (1: 1000; 26 KD; ab59348), Wnt1 (1 μg/mL; 41 KD; ab85060), MMP-9 (1 μg/mL; 95 KD; ab73734), EGFR (1: 1000; 134 KD; ab52894), β-catenin (1: 1000; 92 KD; #9562; Cell signaling technology, Danvers, MA, USA), or β-actin (1: 1000; 45 KD; #4970) overnight at 4°C. The target band was incubated with a goat anti-rabbit IgG H&L (HRP) (1: 5000; ab205718; Abcam, Cambridge, UK) for 2 h. Finally, the signals were detected using SignalFir ECL reagent (#6883) and the gray value of the band was analyzed and calculated using ImageJ (version 5.0, Bio-Rad, Hercules, CA, USA) [17 (link)].
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